Stem cell factor and compositions

ABSTRACT

Novel stem cell factors, oligonucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders involving blood cells are also disclosed.

This application is a continuation of application Ser. No. Continuation07/982,255, filed Nov. 25, 1992 where is hereby incorporated byreference. This is a continuation-in-part application of Ser. No.589,701, filed Oct. 1, 1990, now abandoned, which is acontinuation-in-part application of Ser. No. 573,616 filed Aug. 24,1990, now abandoned, which is a continuation-in-part application of Ser.No. 537,198 filed Jun. 11, 1990, now abandoned, which is acontinuation-in-part application of Ser. No. 422,383 filed Oct. 16,1989, now abandoned, hereby incorporated by reference.

The present invention relates in general to novel factors whichstimulate primitive progenitor cells including early hematopoieticprogenitor cells, and to DNA sequences encoding such factors. Inparticular, the invention relates to these novel factors, to fragmentsand polypeptide analogs thereof and to DNA sequences encoding the same.

BACKGROUND OF THE INVENTION

The human blood-forming (hematopoietic) system is comprised of a varietyof white blood cells (including neutrophils, macrophages, basophils,mast cells, eosinophils, T and B cells), red blood cells (erythrocytes)and clot-forming cells (megakaryocytes, platelets).

It is believed that small amounts of certain hematopoietic growthfactors account for the differentiation of a small number of “stemcells” into a variety of blood cell progenitors for the tremendousproliferation of those cells, and for the ultimate differentiation ofmature blood cells from those lines. The hematopoietic regenerativesystem functions well under normal conditions. However, when stressed bychemotherapy, radiation, or natural myelodysplastic disorders, aresulting period during which patients are seriously leukopenic, anemic,or thrombocytopenic occurs. The development and the use of hematopoieticgrowth factors accelerates bone marrow regeneration during thisdangerous phase.

In certain viral induced disorders, such as acquired autoimmunedeficiency (AIDS) blood elements such as T cells may be specificallydestroyed. Augmentation of T cell production may be therapeutic in suchcases.

Because the hematopoietic growth factors are present in extremely smallamounts, the detection and identification of these factors has reliedupon an array of assays which as yet only distinguish among thedifferent factors on the basis of stimulative effects on cultured cellsunder artificial conditions.

The application of recombinant genetic techniques has clarified theunderstanding of the biological activities of individual growth factors.For example, the amino acid and DNA sequences for human erythropoietin(EPO), which stimulates the production of erythrocytes, have beenobtained. (See, Lin, U.S. Pat. No. 4,703,008, hereby incorporated byreference). Recombinant methods have also been applied to the isolationof cDNA for a human granulocyte colony-stimulating factor, G-CSF (See,Souza, U.S. Pat. No. 4,810,643, hereby incorporated by reference), andhuman granulocyte-macrophage colony stimulating factor (GM-CSF) [Lee, etal., Proc. Natl. Acad. Sci. USA, 82, 4360-4364 (1985); Wong, et al.,Science, 228, 810-814 (1985)], murine G- and GM-CSF [Yokota, et al.,Proc. Natl. Acad. Sci. (USA), 81, 1070 (1984); Fung, et al., Nature,307, 233 (1984); Gough, et al., Nature, 309, 763 (1984)], and humanmacrophage colony-stimulating factor (CSF-1) [Kawasaki, et al., Science,230, 291 (1985)].

The High Proliferative Potential Colony Forming Cell (HPP-CFC) assaysystem tests for the action of factors on early hematopoieticprogenitors [Zont, J. Exp. Med., 159, 679-690 (1984)]. A number ofreports exist in the literature for factors which are active in theHPP-CFC assay. The sources of these factors are indicated in Table 1.The most well characterized factors are discussed below.

An activity in human spleen conditioned medium has been termedsynergistic factor (SF). Several human tissues and human and mouse celllines produce an SF, referred to as SP-1, which synergizes with CSF-1 tostimulate the earliest HPP-CFC. SF-1 has been reported in mediaconditioned by human spleen cells, human placental cells, 5637 cells (abladder carcinoma cell line), and EMT-6 cells (a mouse mammary carcinomacell line). The identity of SF-1 has yet to be determined. Initialreports demonstrate overlapping activities of interleukin-1 with SF-1from cell line 5637 [Zsebo et al., Blood, 71, 962-968 (1988)]. However,additional reports have demonstrated that the combination ofinterleukin-1 (IL-1) plus CSF-1 cannot stimulate the same colonyformation as can be obtained with CSF-1 plus partially purifiedpreparations of 5637 conditioned media [McNiece, Blood, 73, 919 (1989)].

The synergistic factor present in pregnant mouse uterus extract isCSF-1. WEHI-3 cells (murine myelomonocytic leukemia cell line) produce asynergistic factor which appears to be identical to IL-3. Both CSF-1 andIL-3 stimulate hematopoietic progenitors which are more mature than thetarget of SF-1.

Another class of synergistic factor has been shown to be present inconditioned media from TC-1 cells (bone marrow-derived stromal cells).This cell line produces a factor which stimulates both early myeloid andlymphoid cell types. It has been termed hemolymphopoietic growth factor1 (HLGF-1). It has an apparent molecular weight of 120,000 [McNiece etal., Exp. Hematol., 16, 383 (1988)].

Of the known interleukins and CSFs, IL-1, IL-3, and CSF-1 have beenidentified as possessing activity in the HPP-CFC assay. The othersources of synergistic activity mentioned in Table 1 have not beenstructurally identified. Based on the polypeptide sequence andbiological activity profile, the present invention relates to a moleculewhich is distinct from IL-1, IL-3, CSF-1 and SF-1.

TABLE 1 Preparations Containing Factors Active in the HPP-CFC AssaySource ¹ Reference Human Spleen CM [Kriegler, Blood, 60, 503 (1982)]Mouse Spleen CM [Bradley, Exp. Hematol. Today Baum, ed., 285 (1980)] RatSpleen CM [Bradley, supra, (1980)] Mouse lung CM [Bradley, supra,(1980)] Human Placental CM [Kriegler, supra (1982)] Pregnant MouseUterus [Bradley, supra (1980)] GTC-C CM [Bradley, supra (1980)] RH3 CM[Bradley, supra (1980)] PHA PBL [Bradley, supra (1980)] WEHI-3B CM[McNiece, Cell Biol. Int. Rep., 6, 243 (1982)] EMT-6 CM [McNiece, Exp.Hematol., 15, 854 (1987)] L- Cell CM [Kriegler, Exp. Hematol., 12, 844(1984)] 5637 CM [Stanley, Cell, 45, 667 (1986)] TC-1 CM [Song, Blood,66, 273 (1985)] ¹ CM = Conditioned media.

When administered parenterally, proteins are often cleared rapidly fromthe circulation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive proteins may be required to sustaintherapeutic efficacy. Proteins modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified proteins[Abuchowski et al., In: “Enzymes as Drugs”, Holcenberg et al., eds.Wiley-Interscience, New York, N.Y., 367-383 (1981), Newmark et al., J.Appl. Biochem. 4:185-189 (1982), and Katre et al., Proc. Natl. Acad.Sci. USA 84, 1487-1491 (1987)]. Such modifications may also increase theprotein's solubility in aqueous solution, eliminate aggregation, enhancethe physical and chemical stability of the protein, and greatly reducethe immunogenicity and antigenicity of the protein. As a result, thedesired in vivo biological activity may be achieved by theadministration of such polymer-protein adducts less frequently or inlower doses than with the unmodified protein.

Attachment of polyethylene glycol (PEG) to proteins is particularlyuseful because PEG has very low toxicity in mammals [Carpenter et al.,Toxicol. Appl. Pharmacol., 18, 35-40 (1971)]. For example, a PEG adductof adenosine deaminase was approved in the United States for use inhumans for the treatment of severe combined immunodeficiency syndrome. Asecond advantage afforded by the conjugation of PEG is that ofeffectively reducing the immunogenicity and antigenicity of heterologousproteins. For example, a PEG adduct of a human protein might be usefulfor the treatment of disease in other mammalian species without the riskof triggering a severe immune response.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino-terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxyl-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino, hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

It is an object of the present invention to provide a factor causinggrowth of early hematopoietic progenitor cells.

SUMMARY OF THE INVENTION

According to the present invention, novel factors, referred to herein as“stem cell factors” (SCF) having the ability to stimulate growth ofprimitive progenitors including early hematopoietic progenitor cells areprovided. These SCFs also are able to stimulate non-hematopoietic stemcells such as neural stem cells and primordial germ stem cells. Suchfactors include purified naturally-occurring stem cell factors. Theinvention also relates to non-naturally-occurring polypeptides havingamino acid sequences sufficiently duplicative of that ofnaturally-occurring stem cell factor to allow possession of ahematopoietic biological activity of naturally occurring stem cellfactor.

The present invention also provides isolated DNA sequences for use insecuring expression in procaryotic or eukaryotic host cells ofpolypeptide products having amino acid sequences sufficientlyduplicative of that of naturally-occurring stem cell factor to allowpossession of a hematopoietic biological activity of naturally occurringstem cell factor. Such DNA sequences include:

(a) DNA sequences set out in FIGS. 14B, 14C, 15B, 15C, 15D, 42 and 44 ortheir complementary strands;

(b) DNA sequences which hybridize to the DNA sequences defined in (a) orfragments thereof; and p1 (c) DNA sequences which, but for thedegeneracy of the genetic code, would hybridize to the DNA sequencesdefined in (a) and (b).

Also provided are vectors containing such DNA sequences, and host cellstransformed or transfected with such vectors. Also comprehended by theinvention are methods of producing SCF by recombinant techniques, andmethods of treating disorders. Additionally, pharmaceutical compositionsincluding SCF and antibodies specifically binding SCF are provided.

The invention also relates to a process for the efficient recovery ofstem cell factor from a material containing SCF, the process comprisingthe steps of ion exchange chromatographic separation and/or reversephase liquid chromatographic separation.

The present invention also provides a biologically-active adduct havingprolonged in vivo half-life and enhanced potency in mammals, comprisingSCF covalently conjugated to a water-soluble polymer such aspolyethylene glycol or copolymers of polyethylene glycol andpolypropylene glycol, wherein said polymer is unsubstituted orsubstituted at one end with an alkyl group. Another aspect of thisinvention resides in a process for preparing the adduct described above,comprising reacting the SCF with a water-soluble polymer having at leastone terminal reactive group and purifying the resulting adduct toproduce a product with extended circulating half-life and enhancedbiological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an anion exchange chromatogram from the purification ofmammalian SCF.

FIG. 2 is a gel filtration chromatogram from the purification ofmammalian SCF.

FIG. 3 is a wheat germ agglutinin-agarose chromatogram from thepurification of mammalian SCF.

FIG. 4 is a cation exchange chromatogram from the purification ofmammalian SCF.

FIG. 5 is a C₄ chromatogram from the purification of mammalian SCF.

FIG. 6 shows sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) (SDS-PAGE) of C₄ column fractions from FIG. 5.

FIG. 7 is an analytical C₄ chromatogram of mammalian SCF.

FIG. 8 shows SDS-PAGE of C₄ column fractions from FIG. 7.

FIG. 9 shows SDS-PAGE of purified mammalian SCF and deglycosylatedmammalian SCF.

FIG. 10 is an analytical C₄ chromatogram of purified mammalian SCF.

FIG. 11 shows the amino acid sequence (SEQ ID NO:1) of mammalian SCFderived from protein sequencing.

FIG. 12 shows

A. oligonucleotides for rat SCF cDNA (SEQ ID NOS:2-19)

B. oligonucleotides for human SCF DNA (SEQ ID NOS:20-30)

C. universal oligonucleotides (SEQ ID NOS:31-38).

FIG. 13 shows

A. a scheme for polymerase chain reaction (PCR) amplification of rat SCFcDNA

B. a scheme for PCR amplification of human SCF cDNA.

FIG. 14 shows

A. sequencing strategy for rat genomic DNA

B. the nucleic acid sequence of rat (SEQ ID NOS:39 and 40) genomic DNA.

C. the nucleic acid sequence of rat SCF cDNA and amino acid sequence ofrat SCF protein (SEQ ID NOS:41 and 42).

FIG. 15 shows

A. the strategy for sequencing human genomic DNA

B. the nucleic acid sequence of human (SEQ ID NOS:43 and 44) genomic DNA

C. the composite nucleic acid sequence of (SEQ ID NOS:45 and 46) humanSCF cDNA and amino acid sequence of SCF protein.

D. the nucleic acid sequence of genomic DNA and amino acid sequence ofhuman SCF protein, including (SEQ ID NOS:47 and 48) flanking regions andintrons.

FIGS. 16A and B shows the aligned amino acid sequences of human, monkey,dog, mouse, and rat (SEQ ID NOS:49-57) SCF protein.

FIG. 16C shows an elution profile of hSCF¹⁻²⁴⁸ from CHO cells after AspNpeptidase digestion and HPLC.

FIG. 16D shows the nucleotide sequence of the 221 base pair EcoRI-BamHIfragment constructed from (SEQ ID NOS:58 and 59) oligonucleotides thatwere used in creating the plasmid for human [Met⁻¹] SCF¹⁻¹⁶⁵. Uppercaseletters below the nucleotide sequence represent the amino acid sequence.Lowercase letters above the nucleotide sequence show nucleotides in theoriginal hSCF¹⁻¹⁸³ sequence that were altered to generate codons mostfrequently used by E. coli.

FIG. 16E shows the 39 base pair BstEII-BamHI fragment used in creatingthe plasmid for human [Met⁻¹] SCF¹⁻¹⁶⁵ with optimized C-terminal codons.

FIG. 17 shows the structure of mammalian cell expression vector V19.8SCF.

FIG. 18 shows the structure of mammalian CHO cell expression vectorpDSVE.1.

FIG. 19 shows the structure of E. coli expression vector pCFM1156.

FIG. 20 shows

A. a radioimmunoassay of mammalian SCF

B. SDS-PAGE of immune-precipitated mammalian SCF.

FIG. 21 shows Western analysis of recombinant human SCF.

FIG. 22 shows Western analysis of recombinant rat SCF.

FIG. 22A shows radioimmune assay determination of SCF in Human Serum.The percent inhibition of ¹²⁵I-human SCF binding produced was determinedfor various doses of an unlabeled standard of CHO HuSCF¹⁻²⁴⁸ (solidcircles); a sample of NHS Lot 500080713 (open circles); and NHS Lot500081015 (solid triangle).

FIG. 23 is a bar graph showing the effect of COS-1 cell-producedrecombinant rat SCF on bone marrow transplantation.

FIG. 24 shows the effect of recombinant rat SCF on curing the macrocyticanemia of Steel mice.

FIG. 25 shows the peripheral white blood cell count (WBC) of Steel micetreated with recombinant rat SCF.

FIG. 26 shows the platelet counts of Steel mice treated with recombinantrat SCF.

FIG. 27 shows the differential WBC count for Steel mice treated withrecombinant rat SCF¹⁻¹⁶⁴ PEG25.

FIG. 28 shows the lymphocyte subsets for Steel mice treated withrecombinant rat SCF¹⁻¹⁶⁴ PEG25.

FIG. 29 shows the effect of recombinant human sequence SCF treatment ofnormal primates in increasing peripheral WBC count.

FIG. 30 shows the effect of recombinant human sequence SCF treatment ofnormal primates in increasing hematocrits and platelet numbers.

FIG. 31 shows photographs of

A. human bone marrow colonies stimulated by recombinant human SCF¹⁻¹⁶²

B. Wright-Giemsa stained cells from colonies in FIG. 31 A.

FIG. 31C shows proliferation of the UT-7 cell line by E. coli derivedSCFs. Open squares are human [Met⁻¹]SCF¹⁻¹⁶⁴, crosses and open diamondsare human [Met⁻¹]SCF¹⁻¹⁶⁵.

FIG. 32 shows SDS-PAGE of S-Sepharose column fractions from chromatogramshown in FIG. 33

A. with reducing agent

B. without reducing agent.

FIG. 33 is a chromatogram of an S-Sepharose column of E. coli derivedrecombinant human SCF.

FIG. 34 shows SDS-PAGE of C₄ column fractions from chromatogram showingFIG. 35

A. with reducing agent

B. without reducing agent.

FIG. 35 is a chromatogram of a C₄ column of E. coli derived recombinanthuman SCF.

FIG. 36 is a chromatogram of a Q-Sepharose column of CHO derivedrecombinant rat SCF.

FIG. 37 is a chromatogram of a C₄ column of CHO derived recombinant ratSCF.

FIG. 38 shows SDS-PAGE of C₄ column fractions from chromatogram shown inFIG. 37.

FIG. 39 shows SDS-PAGE of purified CHO derived recombinant rat SCFbefore and after de-glycosylation.

FIG. 40 shows

A. gel filtration chromatography of recombinant rat pegylated SCF¹⁻¹⁶⁴reaction mixture

B. gel filtration chromatography of recombinant rat SCF¹⁻¹⁶⁴,unmodified.

FIG. 41 shows labelled SCF binding to fresh leukemic blasts.

FIG. 42 shows human SCF cDNA sequence (SEQ ID NOS:60 and 61) obtainedfrom the HT1080 fibrosarcoma cell line.

FIG. 43 shows an autoradiograph from COS-7 cells expressing humanSCF¹⁻²⁴⁸ and CHO cells expressing human SCF¹⁻¹⁶⁴.

FIG. 44 shows human SCF cDNA sequence (SEQ ID NOS:62 and 63) obtainedfrom the 5637 bladder carcinoma cell line.

FIG. 45 shows the enhanced survival of irradiated mice after SCFtreatment.

FIG. 46 shows the enhanced survival of irradiated mice after bone marrowtransplantation with 5% of a femur and SCF treatment.

FIG. 47 shows the enhanced survival of irradiated mice after bone marrowtransplantation with 0.1 and 20% of a femur and SCF treatment.

FIG. 48 shows radioprotection effects of rat SCF on survival of miceafter irradiation.

FIG. 49 shows radioprotection effects of rat SCF on survival of miceafter irradiation.

FIG. 50 shows a single coinjection of rrSCF plus G-CSF causes anincrease in circulating neutrophils that is approximately additive ascompared to the rrSCF alone- and G-CSF alone-induced neutrophilia. Thekinetics of rrSCF plus G-CSF-induced neutrophilia reflect the combinedeffect of the differing kinetics of rrSCF-induced neutrophilia peakingat 6 hours and G-CSF-induced neutrophilia peaking at 12 hours.

FIG. 51 shows daily coinjection of rrSCF and G-CSF for one week caused ahighly synergistic increase in circulating neutrophils with a markedlinear increase between day 4 and day 6.

FIG. 52 shows a kinetic study of rrSCF plus G-CSF-induced neutrophiliaafter the seventh daily injection shows that the peak of circulatingneutrophils occurs at 12 hours and reaches a level of 69×10³ PMN/mm³.

FIG. 53 shows in vivo administration of SCF-platelet counts.

FIG. 54 shows dose response of rratSCF-PEG on platelet counts.

FIG. 55 shows effect of 5-FU on platelet levels.

FIG. 56 shows 5-FU effect on ACH+ cells in marrow.

FIG. 57 shows mean platelet volume after 5-FU treatment.

FIG. 58 shows SCF mRNA levels after 5-FU treatment. The data in thisfigure were generated from the same marrow samples collected in FIG. 56.Data points are the values determined from individual mice.

FIG. 59 shows the effects of HuSCF and zidovudine on peripheral bloodBFU-E in normal donors. Light density cells were plated in duplicate inthe presence of (A) 1 U/ml or (B) 4 U/ml of erythropoietin, fourconcentrations of zidovudine (0, 10⁻⁷ M, 10⁻⁶ M and 10⁻⁵ M) and fourconcentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). Thebars represent the mean ±S.E.M. for the duplicate determinations of bothnormal donors. All of the increases for HuSCF are statisticallysignificant (independent t-test, 2-tailed, p<O.0l).

FIG. 60 shows the effects of HuSCF and zidovudine on peripheral bloodBFU-E in normal and HIV-infected donors. Light density cells were platedin duplicate in the presence of 1 U/ml of erythropoietin and fourconcentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). Thebars represent the mean for the duplicate determinations.

FIG. 61 shows alteration of the BFU-E ID₅₀ of zidovudine by HuSCF. The50% inhibitory concentration for BFU-E for each level of HuSCF wascalculated as described in the text. The bars represent the mean for thetwo normal donors.

FIG. 62 shows effects of HuSCF on AZT suppression of bone marrow cultureas measured by BFU-E.

FIG. 63 shows effect of HuSCF on AZT suppression of bone marrow cultureas measured by CFU-GM.

FIG. 64 shows effects of HuSCF on gancyclovir suppression of bone marrowculture as measured by BFU-E.

FIG. 65 shows effect of HuSCF on gancyclovir suppression of bone marrowculture as measured by CFU-GM.

FIG. 66 shows effect of rat SCF alone and in combination with CFU-Snumber in a pre-CFU-S assay.

FIG. 67 shows effect of SCF alone and in combination on the recovery ofhemaglobin.

FIG. 68 shows fluorescence emission spectra of human SCF¹⁻¹⁶⁴. Emissionintensity is shown for CHO cell derived [Met⁻¹]SCF¹⁻¹⁶² (dotted line)and E. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (solid line).

FIG. 69 shows circular dichroism of SCF. The far ultraviolet spectra (A)and near ultraviolet spectra (B) are shown for CHO cell-derived[Met⁻¹]SCF¹⁻¹⁶² (dotted line) and E. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (solidline).

FIG. 70 shows second derivative infrared spectra of SCF. The secondderivative infrared spectra in the amide I region (1700-1620 cm⁻¹) forE. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (A) and CHO cell derived [Met⁻¹SCF¹⁻¹⁶²(B) are shown.

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription which provides illustrations of the practice of theinvention in its presently-preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, novel stem cell factors and DNAsequences coding for all or part of such SCFs are provided. The term“stem cell factor” or “SCF” as used herein refers to naturally-occurringSCF (e.g. natural human SCF) as well as non-naturally occurring (i.e.,different from naturally occurring) polypeptides having amino acidsequences and glycosylation sufficiently duplicative of that ofnaturally-occurring stem cell factor to allow possession of ahematopoietic biological activity of naturally-occurring stem cellfactor. Stem cell factor has the ability to stimulate growth of earlyhematopoietic progenitors which are capable of maturing to erythroid,megakaryocyte, granulocyte, lymphocyte, and macrophage cells. SCFtreatment of mammals results in absolute increases in hematopoieticcells of both myeloid and lymphoid lineages. One of the hallmarkcharacteristics of stem cells is their ability to differentiate intoboth myeloid and lymphoid cells [Weissman, Science, 241, 58-62 (1988)].Treatment of Steel mice (Example 8B) with recombinant rat SCF results inincreases of granulocytes, monocytes, erythrocytes, lymphocytes, andplatelets. Treatment of normal primates with recombinant human SCFresults in increases in myeloid and lymphoid cells (Example 8C).

There is embryonic expression of SCF by cells in the migratory pathwayand homing sites of melanoblasts, germ cells, hematopoietic cells, brainand spinal chord.

Early hematopoietic progenitor cells are enriched in bone marrow frommammals which has been treated with 5-Fluorouracil (5-FU). Thechemotherapeutic drug 5-FU selectively depletes late hematopoieticprogenitors. SCF is active on post 5-FU bone marrow.

The biological activity and pattern of tissue distribution of SCFdemonstrates its central role in embryogenesis and hematopoiesis as wellas its capacity for treatment of various stem cell deficiencies.

The present invention provides DNA sequences which include: theincorporation of codons “preferred” for expression by selectednonmammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences which facilitate construction ofreadily-expressed vectors. The present invention also provides DNAsequences coding for polypeptide analogs or derivatives of SCF whichdiffer from naturally-occurring forms in terms of the identity orlocation of one or more amino acid residues (i.e., deletion analogscontaining less than all of the residues specified for SCF; substitutionanalogs, wherein one or more residues specified are replaced by otherresidues; and addition analogs wherein one or more amino acid residuesis added to a terminal or medial portion of the polypeptide) and whichshare some or all the properties of naturally-occurring forms. Thepresent invention specifically provides DNA sequences encoding the fulllength unprocessed amino acid sequence as well as DNA sequences encodingthe processed form of SCF.

Novel DNA sequences of the invention include sequences useful insecuring expression in procaryotic or eucaryotic host cells ofpolypeptide products having at least a part of the primary structuralconformation and one or more of the biological properties ofnaturally-occurring SCF. DNA sequences of the invention specificallycomprise: (a) DNA sequences set forth in FIGS. 14B, 14C, 15B, 15C, 15D,42 and 44 or their complementary strands; (b) DNA sequences whichhybridize (under hybridization conditions disclosed in Example 3 or morestringent conditions) to the DNA sequences in FIGS. 14B, 14C, 15B, 15C,15D, 42, and 44 or to fragments thereof; and (c) DNA sequences which,but for the degeneracy of the genetic code, would hybridize to the DNAsequences in FIGS. 14B, 14C, 15B, 15C, 15D, 42, and 44. Specificallycomprehended in parts (b) and (c) are genomic DNA sequences encodingallelic variant forms of SCF and/or encoding SCF from other mammalianspecies, and manufactured DNA sequences encoding SCF, fragments of SCF,and analogs of SCF. The DNA sequences may incorporate codonsfacilitating transcription and translation of messenger RNA in microbialhosts. Such manufactured sequences may readily be constructed accordingto the methods of Alton et al., PCT published application WO 83/04053.

According to another aspect of the present invention, the DNA sequencesdescribed herein which encode polypeptides having SCF activity arevaluable for the information which they provide concerning the aminoacid sequence of the mammalian protein which have heretofore beenunavailable. The DNA sequences are also valuable as products useful ineffecting the large scale synthesis of SCF by a variety of recombinanttechniques. Put another way, DNA sequences provided by the invention areuseful in generating new and useful viral and circular plasmid DNAvectors, new and useful transformed and transfected procaryotic andeucaryotic host cells (including bacterial and yeast cells and mammaliancells grown in culture), and new and useful methods for cultured growthof such host cells capable of expression of SCF and its relatedproducts.

DNA sequences of the invention are also suitable materials for use aslabeled probes in isolating human genomic DNA encoding SCF and othergenes for related proteins as well as cDNA and genomic DNA sequences ofother mammalian species. DNA sequences may also be useful in variousalternative methods of protein synthesis (e.g., in insect cells) or ingenetic therapy in humans and other mammals. DNA sequences of theinvention are expected to be useful in developing transgenic mammalianspecies which may serve as eucaryotic “hosts” for production of SCF andSCF products in quantity. See, generally, Palmiter et al., Science 222,809-814 (1983).

The present invention provides purified and isolated naturally-occurringSCF (i.e. purified from nature or manufactured such that the primary,secondary and tertiary conformation, and the glycosylation pattern areidentical to naturally-occurring material) as well as non-naturallyoccurring polypeptides having a primary structural conformation (i.e.,continuous sequence of amino acid residues). and glycosylationsufficiently duplicative of that of naturally occurring stem cell factorto allow possession of a hematopoietic biological activity of naturallyoccurring SCF. Such polypetides include derivatives and analogs.

In a preferred embodiment, SCF is characterized by being the product ofprocaryotic or eucaryotic host expression (e.g., by bacterial, yeast,higher plant, insect and mammalian cells in culture) of exogenous DNAsequences obtained by genomic or cDNA cloning or by gene synthesis. Thatis, in a preferred embodiment, SCF is “recombinant SCF.” The products ofexpression in typical yeast (e.g., Saccharomyces cerevisiae) orprocaryote (e.g., E. coli) host cells are free of association with anymammalian proteins. The products of expression in vertebrate [e.g.,non-human mammalian (e.g. COS or CHO) and avian] cells are free ofassociation with any human proteins. Depending upon the host employed,polypeptides of the invention may be glycosylated with mammalian orother eucaryotic carbohydrates or may be non-glycosylated. The host cellcan be altered using techniques such as those described in Lee et al. J.Biol. Chem. 264, 13848 (1989) hereby incorporated by reference.Polypeptides of the invention may also include an initial methionineamino acid residue (at position −1).

In addition to naturally-occurring allelic forms of SCF, the presentinvention also embraces other SCF products such as polypeptide analogsof SCF. Such analogs include fragments of SCF. Following the proceduresof the above-noted published application by Alton et al. (WO 83/04053),one can readily design and manufacture genes coding for microbialexpression of polypeptides having primary conformations which differfrom that herein specified for in terms of the identity or location ofone or more residues (e.g., substitutions, terminal and intermediateadditions and deletions). Alternately, modifications of cDNA and genomicgenes can be readily accomplished by well-known site-directedmutagenesis techniques and employed to generate analogs and derivativesof SCF. Such products share at least one of the biological properties ofSCF but may differ in others. As examples, products of the inventioninclude those which are foreshortened by e.g., deletions; or those whichare more stable to hydrolysis (and, therefore, may have more pronouncedor longer-lasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by, e.g., alanine or serineresidues and are potentially more easily isolated in active form frommicrobial systems; or which have one or more tyrosine residues replacedby phenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within SCF, which fragments may possess oneproperty of SCF (e.g., receptor binding) and not others (e.g., earlyhematopoietic cell growth activity). It is noteworthy that activity isnot necessary for any one or more of the products of the invention tohave therapeutic utility [see, Weiland et al., Blut, 44, 173-175 (1982)]or utility in other contexts, such as in assays of SCF antagonism.Competitive antagonists may be quite useful in, for example, cases ofoverproduction of SCF or cases of human leukemias where the malignantcells overexpress receptors for SCF, as indicated by the overexpressionof SCF receptors in leukemic blasts (Example 13).

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence extant in naturally-occurringproteins, glycoproteins and nucleoproteins. More specifically,relatively low molecular weight polypeptides have been shown toparticipate in immune reactions which are similar in duration and extentto the immune reactions of physiologically-significant proteins such asviral antigens, polypeptide hormones, and the like. Included among theimmune reactions of such polypeptides is the provocation of theformation of specific antibodies in immunologically-active animals[Lerner et al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294,654-656 (1981); Walter et al., Proc. Natl. Acad. Sci. USA, 77, 5197-5200(1980); Lerner et al., Proc. Natl. Acad. Sci. USA, 78, 3403-3407 (1981);Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886 (1981); Wong etal., Proc. Natl. Acad. Sci. USA, 79, 5322-5326 (1982); Baron et al.,Cell, 28, 395-404 (1982); Dressman et al., Nature, 295, 185-160 (1982);and Lerner, Scientific American, 248, 66-74 (1983)]. See, also, Kaiseret al. [Science, 223, 249-255 (1984)] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The present invention also includes that class of polypeptides coded forby portions of the DNA complementary to the protein-coding strand of thehuman cDNA or genomic DNA sequences of SCF, i.e., “complementaryinverted proteins” as described by Tramontano et al. [Nucleic Acid Res.,12, 5049-5059 (1984)].

Representative SCF polypeptides of the present invention include but arenot limited to SCF¹⁻¹⁴⁸, SCF¹⁻¹⁶², SCF¹⁻¹⁶⁴, SCF¹⁻¹⁶⁵ and SCF¹⁻¹⁸³ inFIG. 15C; SCF¹⁻¹⁸⁵, SCF¹⁻¹⁸⁸, SCF¹⁻¹⁸⁹ and SCF¹⁻²⁴⁸ in FIG. 42; andSCF¹⁻¹⁵⁷, SCF¹⁻¹⁶⁰, SCF¹⁻¹⁶¹ and SCF¹⁻²²⁰ in FIG. 44.

SCF can be purified using techniques known to those skilled in the art.The subject invention comprises a method of purifying SCF from an SCFcontaining material such as conditioned media or human urine, serum, themethod comprising one or more of steps such as the following: subjectingthe SCF containing material to ion exchange chromatography (eithercation or anion exchange chromatography); subjecting the SCF containingmaterial to reverse phase liquid chromatographic separation involving,for example, an immobilized C₄ or C₆ resin; subjecting the fluid toimmobilized-lectin chromatography, i.e., binding of SCF to theimmobilized lectin, and eluting with the use of a sugar that competesfor this binding. Details in the use of these methods will be apparentfrom the descriptions given in Examples 1, 10, and 11 for thepurification of SCF. The techniques described in Example 2 of the Lai etal. U.S. Pat. No. 4,667,016, hereby incorporated by reference are alsouseful in purifying stem cell factor.

Isoforms of SCF are isolated using standard techniques such as thetechniques set forth in commonly owned U.S. Ser. No. 421,444 entitledErythropoietin Isoforms, filed Oct. 13, 1989, hereby incorporated byreference.

Also comprehended by the invention are pharmaceutical compositionscomprising therapeutically effective amounts of polypeptide products ofthe invention together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers useful in SCFtherapy. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. Such compositions are liquids or lyophilizedor otherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent adsorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycol), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), bulking substances or tonicitymodifiers (e.g., lactose, mannitol), covalent attachment of polymerssuch as polyethylene glycol to the protein (described in Example 12below), complexation with metal ions, or incorporation of the materialinto or onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc. or into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance of SCF. The choice of composition will dependon the physical and chemical properties of the protein having SCFactivity. For example, a product derived from a membrane-bound form ofSCF may require a formulation containing detergent. Controlled orsustained release compositions include formulation in lipophilic depots(e.g., fatty acids, waxes, oils). Also comprehended by the invention areparticulate compositions coated with polymers (e.g., poloxamers orpoloxamines) and SCF coupled to antibodies directed againsttissue-specific receptors, ligands or antigens or coupled to ligands oftissue-specific receptors. Other embodiments of the compositions of theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,including parenteral, pulmonary, nasal and oral.

The invention also comprises compositions including one or moreadditional hematopoietic factors such as EPO, G-CSF, GM-CSF, CSF-1,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IGF-I, or LIF (Leukemic Inhibitory Factor).

Polypeptides of the invention may be “labeled” by association with adetectable marker substance (e.g., radiolabeled with ¹²⁵I orbiotinylated) to provide reagents useful in detection and quantificationof SCF or its receptor bearing cells in solid tissue and fluid samplessuch as blood or urine.

Biotinylated SCF is useful in conjunction with immobilized streptavidinto purge leukemic blasts from bone marrow in autologous bone marrowtransplantation. Biotinylated SCF is useful in conjunction withimmobilized streptavidin to enrich for stem cells in autologous orallogeneic stem cells in autologous or allogeneic bone marrowtransplantation. Toxin conjugates of SCF, such as ricin [Uhr, Prog.Clin. Biol. Res. 288, 403-412 (1989)] diptheria toxin [Moolten, J. Natl.Con. Inst., 55, 473-477 (1975)], and radioisotopes are useful for directanti-neoplastic therapy (Example 13) or as a conditioning regimen forbone marow transplantation.

Nucleic acid products of the invention are useful when labeled withdetectable markers (such as radiolabels and non-isotopic labels such asbiotin) and employed in hybridization processes to locate the human SCFgene position and/or the position of any related gene family in achromosomal map. They are also useful for identifying human SCF genedisorders at the DNA level and used as gene markers for identifyingneighboring genes and their disorders. The human SCF gene is encoded onchromosome 12, and the murine SCF gene maps to chromosome 10 at the S1locus.

SCF is useful, alone or in combination with other therapy, in thetreatment of a number of hematopoietic disorders. SCF can be used aloneor with one or more additional hematopoietic factors such as EPO, G-CSF,GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-1, IGF-I or LIF in the treatment of hematopoieticdisorders.

There is a group of stem cell disorders which are characterized by areduction in functional marrow mass due to toxic, radiant, orimmunologic injury and which may be treatable with SCF. Aplastic anemiais a stem cell disorder in which there is a fatty replacement ofhematopoietic tissue and pancytopenia. SCF enhances hematopoieticproliferation and is useful in treating aplastic anemia (Example 8B).Steel mice are used as a model of human aplastic anemia [Jones, Exp.Hematol., 11, 571-580 (1983)]. Promising results have been obtained withthe use of a related cytokine, GM-CSF in the treatment of aplasticanemia [Antin, et al., Blood, 70, 129a (1987)]. Paroxysmal nocturnalhemoglobinuria (PNH) is a stem cell disorder characterized by formationof defective platelets and granulocytes as well as abnormalerythrocytes.

There are many diseases which are treatable with SCF. These include thefollowing: myelofibrosis, myelosclerosis, osteopetrosis, metastaticcarcinoma, acute leukemia, multiple myeloma, Hodgkin's disease,lymphoma, Gaucher's disease, Niemann-Pick disease, Letterer-Siwedisease, refractory erythroblastic anemia, Di Guglielmo syndrome,congestive splenomegaly, Hodgkin's disease, Kala azar, sarcoidosis,primary splenic pancytopenia, miliary tuberculosis, disseminated fungusdisease, Fulminating septicemia, malaria, vitamin B₁₂ and folic aciddeficiency, pyridoxine deficiency, Diamond Blackfan anemia,hypopigmentation disorders such as piebaldism and vitiligo. Theerythroid, megakaryocyte, and granulocytic stimulatory properties of SCFare illustrated in Example 8B and 8C.

Enhancement of growth in non-hematopoietic stem cells such as primordialgerm cells, neural crest derived melanocytes, commissural axonsoriginating from the dorsal spinal cord, crypt cells of the gut,mesonephric and metanephric kidney tubules, and olfactory bulbs is ofbenefit in states where specific tissue damage has occurred to thesesites. SCF is useful for treating neurological damage and is a growthfactor for nerve cells. SCF is useful during in vitro fertilizationprocedures or in treatment of infertility states. SCF is useful fortreating intestinal damage resulting from irradiation or chemotherapy.

There are stem cell myeloproliferative disorders such as polycythemiavera, chronic myelogenous leukemia, myeloid mataplasia, primarythrombocythemia, and acute leukemias which are treatable with SCF,anti-SCF antibodies, or SCF-toxin conjugates.

There are numerous cases which document the increased proliferation ofleukemic cells to the hematopoietic cell growth factors G-CSF, GM-CSF,and IL-3 [Delwel, et al., Blood, 72, 1944-1949 (1988)]. Since thesuccess of many chemotherapeutic drugs depends on the fact thatneoplastic cells cycle more actively than normal cells, SCF alone or incombination with other factors acts as a growth factor for neoplasticcells and sensitizes them to the toxic effects of chemotherapeuticdrugs. The overexpression of SCF receptors on leukemic blasts is shownin Example 13.

A number of recombinant hematopoietic factors are undergoinginvestigation for their ability to shorten the leukocyte nadir resultingfrom chemotherapy and radiation regimens. Although these factors arevery useful in this setting, there is an early hematopoietic compartmentwhich is damaged, especially by radiation, and has to be repopulatedbefore these later-acting growth factors can exert their optimal action.The use of SCF alone or in combination with these factors furthershortens or eliminates the leukocyte and platelet nadir resulting fromchemotherapy or radiation treatment. In addition, SCF allows for a doseintensification of the anti-neoplastic or irradiation regimen (Example19).

SCF is useful for expanding early hematopoietic progenitors insyngeneic, allogeneic, or autologous bone marrow transplantation. Theuse of hematopoietic growth factors has been shown to decrease the timefor neutrophil recovery after transplantation [Donahue, et al., Nature,321, 872-875 (1986) and Welte et al., J. Exp. Med., 165, 941-948,(1987)]. For bone marrow transplantation, the following three scenariosare used alone or in combination: a donor is treated with SCF alone orin combination with other hematopoietic factors prior to bone marrowaspiration or peripheral blood leucophoresis to increase the number ofcells available for transplantation; the bone marrow is treated in vitroto activate or expand the cell number prior to transplantation; finally,the recipient is treated to enhance engraftment of the donor marrow.

SCF is useful for enhancing the efficiency of gene therapy based ontransfecting (or infecting with a retroviral vector) hematopoietic stemcells. SCF permits culturing and multiplication of the earlyhematopoietic progenitor cells which are to be transfected. The culturecan be done with SCF alone or in combination with IL-6, IL-3, or both.Once tranfected, these cells are then infused in a bone marrowtransplant into patients suffering from genetic disorders. [Lim, Proc.Natl. Acad. Sci, 86, 8892-8896 (1989)]. Examples of genes which areuseful in treating genetic disorders include adenosine deaminase,glucocerebrosidase, hemoglobin, and cystic fibrosis.

SCF is useful for treatment of acquired immune deficiency (AIDS) orsevere combined immunodeficiency states (SCID) alone or in combinationwith other factors such as IL-7 (see Example 14). Illustrative of thiseffect is the ability of SCF therapy to increase the absolute level ofcirculating T-helper (CD4+, OKT₄+) lymphocytes. These cells are theprimary cellular target of human immunodeficiency virus (HIV) leading tothe immunodeficiency state in AIDS patients [Montagnier, in Human T-CellLeukemia/Lymphoma Virus, ed. R. C. Gallo, Cold Spring Harbor, N.Y.,369-379 (1984)]. In addition, SCF is useful for combatting themyelosuppressive effects of anti-HIV drugs such as AZT [Gogu LifeSciences, 45, No. 4 (1989)].

SCF is useful for enhancing hematopoietic recovery after acute bloodloss.

In vivo treatment with SCF is useful as a boost to the immune system forfighting neoplasia (cancer). An example of the therapeutic utility ofdirect immune function enhancement by a recently cloned cytokine (IL-2)is described in Rosenberg et al., N. Eng. J. Med., 313 1485 (1987).

The administration of SCF with other agents such as one or more otherhematopoietic factors, is temporally spaced or given together. Priortreatment with SCF enlarges a progenitor population which responds toterminally-acting hematopoietic factors such as G-CSF or EPO. The routeof administration may be intravenous, intraperitoneal sub-cutaneous, orintramuscular.

The subject invention also relates to antibodies specifically bindingstem cell factor. Example 7 below describes the production of polyclonalantibodies. A further embodiment of the invention is monoclonalantibodies specifically binding SCF (see Example 20). In contrast toconventional antibody (polyclonal) preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Monoclonal antibodies are useful to improve the selectivity andspecificity of diagnostic and analytical assay methods usingantigen-antibody binding. Also, they are used to neutralize or removeSCF from serum. A second advantage of monoclonal antibodies is that theycan be synthesized by hybridoma cells in culture, uncontaminated byother immunoglobulins. Monoclonal antibodies may be prepared fromsupernatants of cultured hybridoma cells or from ascites induced byintraperitoneal inoculation of hybridoma cells into mice. The hybridomatechnique described originally by Köhler and Milstein [Eur. J. Immunol.6, 511-519 (1976)] has been widely applied to produce hybrid cell linesthat secrete high levels of monoclonal antibodies against many specificantigens.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1 Purification/Characterization of Stem Cell Factor from BuffaloRat Liver Cell Conditoned Medium

A. In Vitro Biological Assays

1. HPP-CFC Assay

There are a variety of biological activities which can be attributed tothe natural mammalian rat SCF as well as the recombinant rat SCFprotein. One such activity is its effect on early hematopoietic cells.This activity can be measured in a High Proliferative Potential ColonyForming Cell (HPP-CFC) assay [Zsebo, et al., supra (1988)]. Toinvestigate the effects of factors on early hematopoietic cells, theHPP-CFC assay system utilizes mouse bone marrow derived from animals 2days after 5-fluorouracil (5-FU) treatment. The chemotherapeutic drug5-FU selectively depletes late hematopoietic progenitors, allowing fordetection of early progenitor cells and hence factors which act on suchcells. The rat SCF is plated in the presence of CSF-1 or IL-6 insemi-solid agar cultures. The agar cultures contain McCoys completemedium (GIBCO), 20% fetal bovine serum, 0.3% agar, and 2×10⁵ bone marrowcells/ml. The McCoys complete medium contains the following components:1×McCoys medium supplemented with 0.1 mM pyruvate, 0.24× essential aminoacids, 0.24× non-essential amino acids, 0.027% sodium bicarbonate, 0.24×vitamins, 0.72 mM glutamine, 25 μg/ml L-serine, and 12 μg/mlL-asparagine. The bone marrow cells are obtained from Balb/c miceinjected i.v. with 150 mg/kg 5-FU. The femurs are harvested 2 days post5-FU treatment of the mice and bone marrow is flushed out. The red bloodcells are lysed with red blood cell lysing reagent (Becton Dickenson)prior to plating. Test substances are plated with the above mixture in30 mm dishes. Fourteen days later the colonies (>1 mm in diameter) whichcontain thousands of cells are scored. This assay was used throughoutthe purification of natural mammalian cell-derived rat SCF.

In a typical assay, rat SCF causes the proliferation of approximately 50HPP-CFC per 200,000 cells plated. The rat SCF has a synergistic activityon 5-FU treated mouse bone marrow cells; HPP-CFC colonies will not formin the presence of single factors but the combination of SCF and CSF-1or SCF and IL-6 is active in this assay.

2. MC/9 Assay

Another useful biological activity of both naturally-derived andrecombinant rat SCF is the ability to cause the proliferation of theIL-4 dependent murine mast cell line, MC/9 (ATCC CRL 8306). MC/9 cellsare cultured with a source of IL-4 according to the ATCC CRL 8306protocol. The medium used in the bioassay is RPMI 1640, 4% fetal bovineserum, 5×10⁵M 2-mercaptoethanol, and 1× glutamine-pen-strep. The MC/9cells proliferate in response to SCF without the requirement for othergrowth factors. This proliferation is measured by first culturing thecells for 24 h without growth factors, plating 5000 cells in each wellof 96 well plates with test sample for 48 h, pulsing for 4 h with 0.5uCi ³H-thymidine (specific activity 20 Ci/mmol), harvesting the solutiononto glass fiber filters, and then measuring specifically-boundradioactivity. This assay was used in the purification of mammalian cellderived rat SCF after the ACA 54 gel filtration step, section C2 of thisExample. Typically, SCF caused a 4-10 fold increase in CPM overbackground.

3. CFU-GM

The action of purified mammalian SCF, both naturally-derived andrecombinant, free from interfering colony stimulating factors (CSFs), onnormal undepleted mouse bone marrow has been ascertained. A CFU-GM assay[Broxmeyer et al. Exp. Hematol., 5, 87 (1977)] is used to evaluate theeffect of SCF on normal marrow. Briefly, total bone marrow cells afterlysis of red blood cells are plated in semi-solid agar culturescontaining the test substance. After 10 days, the colonies containingclusters of >40 cells are scored. The agar cultures can be dried downonto glass slides and the morphology of the cells can be determined viaspecific histological stains.

On normal mouse bone marrow, the purified mammalian rat SCF was apluripotential CSF, stimulating the growth of colonies consisting ofimmature cells, neutrophils, macrophages, eosinophils, andmegakaryocytes without the requirement for other factors. From 200,000cells plated, over 100 such colonies grow over a 10 day period. Both ratand human recombinant SCF stimulate the production of erythroid cells incombination with EPO, see Example 9.

B. Conditioned Medium

Buffalo rat liver (BRL) 3A cells, from the American Type CultureCollection (ATCC CRL 1442), were grown on microcarriers in a 20 literperfusion culture system for the production of SCF. This system utilizesa Biolafitte fermenter (Model ICC-20) except for the screens used forretention of microcarriers and the oxygenation tubing. The 75 micronmesh screens are kept free of microcarrier clogging by periodic backflushing achieved through a system of check valves andcomputer-controlled pumps. Each screen alternately acts as medium feedand harvest screen. This oscillating flow pattern ensures that thescreens do not clog. Oxygenation was provided through a coil of siliconetubing (50 feet long, 0.25 inch ID, 0.03 inch wall). The growth mediumused for the culture of BRL 3A cells was Minimal Essential Medium (withEarle's Salts) (GIBCO), 2 mM glutamine, 3 g/L glucose, tryptosephosphate (2.95 g/L), 5% fetal bovine serum and 5% fetal calf serum. Theharvest medium was identical except for the omission of serum. Thereactor contained Cytodex 2 microcarriers (Pharmacia) at a concentrationof 5 g/L and was seeded with 3×10⁹ BRL 3A cells grown in roller bottlesand removed by trypsinization. The cells were allowed to attach to andgrow on the microcarriers for eight days. Growth medium was perfusedthrough the reactor as needed based on glucose consumption. The glucoseconcentration was maintained at approximately 1.5 g/L. After eight days,the reactor was perfused with six volumes of serum free medium to removemost of the serum (protein concentration <50 ug/ml). The reactor wasthen operated batchwise until the glucose concentration fell below 2g/L. From this point onward, the reactor was operated at a continuousperfusion rate of approximately 10 L/day. The pH of the culture wasmaintained at 6.9±0.3 by adjusting the CO₂ flow rate. The dissolvedoxygen was maintained higher than 20% of air saturation by supplementingwith pure oxygen as necessary. The temperature was maintained at 37±0.5°C.

Approximately 336 liters of serum free conditioned medium was generatedfrom the above system and was used as the starting material for thepurification of natural mammalian cell-derived rat SCF.

20 C. Purification

All purification work was carried out at 4° C. unless indicatedotherwise.

1. DEAE-cellulose Anion Exchange Chromatography Conditioned mediumgenerated by serum-free growth of BRL 3A cells was clarified byfiltration through 0.45μ Sartocapsules (Sartorius). Several differentbatches (41 L, 27 L, 39 L, 30.2 L, 37.5 L, and 161 L) were separatelysubjected to concentration, diafiltration/buffer exchange, andDEAE-cellulose anion exchange chromatography, in similar fashion foreach batch. The DEAE-cellulose pools were then combined and processedfurther as one batch in sections C2-5 of this Example. To illustrate,the handling of the 41 L batch was as follows. The filtered conditionedmedium was concentrated to ˜700 ml using a Millipore Pellicon tangentialflow ultrafiltration apparatus with four 10,000 molecular weight cutoffpolysulfone membrane cassettes (20 ft² total membrane area; pump rate˜1095 ml/min and filtration rate 250-315 ml/min). Diafiltration/bufferexchange in preparation for anion exchange chromatography was thenaccomplished by adding 500 ml of 50 mM Tris-HCl, pH 7.8 to theconcentrate, reconcentrating to 500 ml using the tangential flowultrafiltration apparatus, and repeating this six additional times. Theconcentrated/diafiltered preparation was finally recovered in a volumeof 700 ml. The preparation was applied to a DEAE-cellulose anionexchange column (5×20.4 cm; Whatman DE-52 resin) which had beenequilibrated with the 50 mM Tris-HCl, pH 7.8 buffer. After sampleapplication, the column was washed with 2050 ml of the Tris-HCl buffer,and a salt gradient (0-300 mM NaCl in the Tris-HCl buffer; 4 L totalvolume) was applied. Fractions of 15 ml were collected at a flow rate of167 ml/h. The chromatography is shown in FIG. 1. HPP-CFC colony numberrefers to biological activity in the HPP-CFC assay; 100 μl from theindicated fractions was assayed. Fractions collected during the sampleapplication and wash are not shown in the Figure; no biological activitywas detected in these fractions.

The behavior of all conditioned media batches subjected to theconcentration, diafiltration/buffer exchange, and anion exchangechromatography was similar. Protein concentrations for the batches,determined by the method of Bradford [Anal. Biochem. 72, 248-254 (1976)]with bovine serum albumin as standard were in the range 30-50 μg/ml. Thetotal volume of conditioned medium utilized for this preparation wasabout 336 L.

2. ACA 54 Gel Filtration Chromatography

Fractions having biological activity from the DEAE-cellulose columns runfor each of the six conditioned media batches referred to above (forexample, fractions 87-114 for the run shown in FIG. 1) were combined(total volume 2900 ml) and concentrated to a final volume of 74 ml withthe use of Amicon stirred cells and YM10 membranes. This material wasapplied to an ACA 54 (LKB) gel filtration column (FIG. 2) equilibratedin 50 mM Tris-HCl, 50 mM NaCl, pH 7.4. Fractions of 14 ml were collectedat a flow rate of 70 ml/h. Due to inhibitory factors co-eluting with theactive fractions, the peak of activity (HPP-CFC colony number) appearssplit; however, based on previous chromatograms, the activity co-eluteswith the major protein peak and therefore one pool of the fractions wasmade.

3. Wheat Germ Agglutinin-Agarose Chromatography

Fractions 70-112 from the ACA 54 gel filtration column were pooled (500ml). The pool was divided in half and each half subjected tochromatography using a wheat germ agglutinin-agarose column (5×24.5 cm;resin from E-Y Laboratories, San Mateo, Calif.; wheat germ agglutininrecognizes certain carbohydrate structures) equilibrated in 20 mMTris-HCl, 500 mM NaCl, pH 7.4. After the sample applications, the columnwas washed with about 2200 ml of the column buffer, and elution of boundmaterial was then accomplished by applying a solution of 350 mMN-acetyl-D-glucosamine dissolved in the column buffer, beginning atfraction ˜210 in FIG. 3. Fractions of 13.25 ml were collected at a flowrate of 122 ml/h. One of the chromatographic runs is shown in FIG. 3.Portions of the fractions to be assayed were dialyzed againstphosphate-buffered saline; 5 ul of the dialyzed materials were placedinto the MC/9 assay (cpm values in FIG. 3) and 10 μl into the HPP-CFCassay (colony number values in FIG. 3). It can be seen that the activematerial bound to the column and was eluted with theN-acetyl-D-glucosamine, whereas much of the contaminating materialpassed through the column during sample application and wash.

4. S-Sepharose Fast Flow Cation Exchange Chromatography

Fractions 211-225 from the wheat germ agglutinin-agarose chromatographyshown in FIG. 3 and equivalent fractions from the second run were pooled(375 ml). and dialyzed against 25 mM sodium formate, pH 4.2. To minimizethe time of exposure to low pH, the dialysis was done over a period of 8h, against 5 L of buffer, with four changes being made during the 8 hperiod. At the end of this dialysis period, the sample volume was 480 mland the pH and conductivity of the sample were close to those of thedialysis buffer. Precipitated material appeared in the sample duringdialysis. This was removed by centrifugation at 22,000×g for 30 min, andthe supernatant from the centrifuged sample was applied to a S-SepharoseFast Flow cation exchange column (3.3×10.25 cm; resin from Pharmacia)which had been equilibrated in the sodium formate buffer. Flow rate was465 ml/h and fractions of 14.2 ml were collected. After sampleapplication, the column was washed with 240 ml of column buffer andelution of bound material was carried out by applying a gradient of0-750 mM NaCl (NaCl dissolved in column buffer; total gradient volume2200 ml), beginning at fraction ˜45 in FIG. 4. The elution profile isshown in FIG. 4. Collected fractions were adjusted to pH 7-7.4 byaddition of 200 μl of 0.97 M Tris base. The cpm in FIG. 4 again refer tothe results obtained in the MC/9 biological assay; portions of theindicated fractions were dialyzed against phosphate-buffered saline, and20 μl placed into the assay. It can be seen in FIG. 4 that the majorityof biologically active material passed through the column unbound,whereas much of the contaminating material bound and was eluted in thesalt gradient.

5. Chromatography Using Silica-Bound Hydrocarbon Resin

Fractions 4-40 from the S-Sepharose column of FIG. 4 were pooled (540ml). 450 ml of the pool was combined with an equal volume of buffer B(100 mM ammonium acetate, pH 6:isopropanol; 25:75) and applied at a flowrate of 540 ml/h to a C₄ column (Vydac Proteins C₄; 2.4×2 cm)equilibrated with buffer A (60 mM ammonium acetate, pH 6:isopropanol;62.5:37.5). After sample application, the flow rate was reduced to 154ml/h and the column was washed with 200 ml of buffer A. A lineargradient from buffer A to buffer B (total volume 140 ml) was thenapplied, and fractions of 9.1 ml were collected. Portions of the poolfrom S-Sepharose chromatography, the C₄ column starting sample,runthrough pool, and wash pool were brought to 40 μg/ml bovine serumalbumin by addition of an appropriate volume of a 1 mg/ml stocksolution, and dialyzed against phosphate-buffered saline in preparationfor biological assay. Similarly, 40 μl aliquots of the gradientfractions were combined with 360 μl of phosphate-buffered salinecontaining 16 μg bovine serum albumin, and this was followed by dialysisagainst phosphate-buffered saline in preparation for biological assay.These various fractions were assayed by the MC/9 assay (6.3 μl aliquotsof the prepared gradient fractions; cpm in FIG. 5). The assay resultsalso indicated that about 75% of the recovered activity was in therunthrough and wash fractions, and 25% in the gradient fractionsindicated in FIG. 5. SDS-PAGE [Laemmli, Nature, 227, 680-685 (1970);stacking gels contained 4% (w/v) acrylamide and separating gelscontained 12.5% (w/v) acrylamide] of aliquots of various fractions isshown in FIG. 6. For the gel shown, sample aliquots were dried undervacuum and then redissolved using 20 μl sample treatment buffer(nonreducing, i.e., without 2-mercaptoethanol) and boiled for 5 minprior to loading onto the gel. Lanes A and B represent column startingmaterial (75 μl out of 890 ml) and column runthrough (75 μl out of 880ml), respectively; the numbered marks at the left of the Figurerepresent migration positions (reduced) of markers having molecularweights of 10³ times the indicated numbers, where the markers arephosphorylase b (M_(r) of 97,400), bovine serum albumin (M_(r) of66,200), ovalbumin (M_(r) of 42,700), carbonic anhydrase (M_(r) of31,000), soybean trypsin inhibitor (M_(r) of 21,500), and lysozyme(M_(r) of 14,400); lanes 4-9 represent the corresponding fractionscollected during application of the gradient (60 μl out of 9.1 ml). Thegel was silver-stained [Morrissey, Anal. Biochem., 117, 307-310 (1981)].It can be seen by comparing lanes A and B that the majority of stainablematerial passes through the column. The stained material in fractions4-6 in the regions just above and below the M_(r) 31,000 standardposition coincides with the biological activity detected in the gradientfractions (FIG. 5) and represents the biologically active material. Itshould be noted that this material is visualized in lanes 4-6, but notin lanes A and/or B, because a much larger proportion of the totalvolume (0.66% of the total for fractions 4-6 versus 0.0084% of the totalfor lanes A and B) was loaded for the former. Fractions 4-6 from thiscolumn were pooled.

As mentioned above, roughly 75% of the recovered activity ran throughthe C₄ column of FIG. 5. This material was rechromatographed using C₄resin essentially as described above, except that a larger column(1.4×7.8 cm) and slower flow rate (50-60 ml/h throughout) were used.Roughly 50% of recovered activity was in the runthrough, and 50% ingradient fractions showing similar appearance on SDS-PAGE to that of theactive gradient fractions in FIG. 6. Active fractions were pooled (29ml).

An analytical C₄ column was also performed essentially as stated aboveand the fractions were assayed in both bioassays. As indicated in FIG. 7of the fractions from this analytical column, both the MC/9 and HPP-CFCbioactivities co-elute. SDS-PAGE analysis (FIG. 8) reveals the presenceof the M_(r) ˜31,000 protein in the column fractions which containbiological activity in both assays.

Active material in the second (relatively minor) activity peak seen inS-Sepharose chromatography (e.g. FIG. 4, fractions 62-72, earlyfractions in the salt gradient) has also been purified by C₄chromatography. It exhibited the same behavior on SDS-PAGE and had thesame N-terminal amino acid sequence (see Example 2D) as the materialobtained by C₄ chromatography of the S-Sepharose runthrough fractions.

6. Purification Summary

A summary of the purification steps described in 1-5 above is given inTable 2.

TABLE 2 Summary of Purification of Mammalian SCF Total Step Volume (ml)Protein (mg)⁵ Conditioned medium 335,700 13,475 DEAE cellulose¹  2,900 2,164 ACA-54    550  1,513 Wheat germ agglutinin-agarose²    375   431S-Sepharose    540⁴    10 C₄ resin³    57.3 0.25-0.40⁶ ¹Values givenrepresent sums of the values for the different batches described in thetext. ²As described above in this Example, precipitated material whichappeared during dialysis of this sample in preparation for S-Sepharosechromatography was removed by centrifugation. The sample aftercentrifugation (480 ml) contained 264 mg of total protein. ³Combinationof the active gradient fractions from the two C₄ columns run in sequenceas described. ⁴Only 450 ml of this material was used for the followingstep (this Example, above). ⁵Determined by the method of Bradford(supra, 1976) except where indicated otherwise. ⁶Estimate, based onintensity of silver-staining after SDS-PAGE, and on amino acidcomposition analysis as described in section K of Example 2.

D. SDS-PAGE and Glycosidase Treatments

SDS-PAGE of pooled gradient fractions from the two large scale C₄ columnruns are shown in FIG. 9. Sixty μl of the pool for the first C₄ columnwas loaded (lane 1), and 40 μl of the pool for the second C₄ column(lane 2). These gel lanes were silver-stained. Molecular weight markerswere as described for FIG. 6. As mentioned, the diffusely-migratingmaterial above and below the M_(r) 31,000 marker position represents thebiologically active material; the apparent heterogeneity is largely dueto heterogeneity in glycosylation.

To characterize the glycosylation, purified material was iodinated with¹²⁵I, treated with a variety of glycosidases, and analyzed by SDS-PAGE(reducing conditions) with autoradiography. Results are shown in FIG. 9.Lanes 3 and 9, ¹²⁵I-labeled material without any glycosidase treatment.Lanes 4-8 represent ¹²⁵I-labeled material treated with glycosidases, asfollows. Lane 4, neuraminidase. Lane 5, neuraminidase and O-glycanase.Lane 6, N-glycanase. Lane 7, neuraminidase and N-glycanase. Lane 8,neuraminidase, O-glycanase, and N-glycanase. Conditions were 5 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 33 mM2-mercaptoethanol, 10 mM Tris-HCl, pH 7-7.2, for 3 h at 37° C.Neuraminidase (from Arthrobacter ureafaciens; Calbiochem) was used at0.23 units/ml final concentration. O-Glycanase (Genzyme;endo-alpha-N-acetyl-galactosaminidase) was used at 45 milliunits/ml.N-Glycanase (Genzyme; peptide:N-glycosidase F;peptide-N⁴[N-acetyl-beta-glucosaminyl]asparagine amidase) was used at 10units/ml.

Similar results to those of FIG. 9 were obtained upon treatment ofunlabeled purified SCF with glycosidases, and visualization of productsby silver-staining after SDS-PAGE.

Where appropriate, various control incubations were carried out. Theseincluded: incubation in appropriate buffer, but without glycosidases, toverify that results were due to the glycosidase preparations added;incubation with glycosylated proteins (e.g. glycosylated recombinanthuman erythropoietin) known to be substrates for the glycosidases, toverify that the glycosidase enzymes used were active; and incubationwith glycosidases but no substrate, to verify that the glycosidases werenot themselves contributing to or obscuring the visualized gel bands.

Glycosidase treatments were also carried out withendo-beta-N-acetylglucosamidase F (endo F; NEN Dupont) and withendo-beta-N-acetylglucosaminidase H (endo H; NEN Dupont), again withappropriate control incubations. Conditions of treatment with endo Fwere: boiling 3 min in the presence of 1% (w/v) SDS, 100 mM2-mercaptoethanol, 100 mM EDTA, 320 mM sodium phosphate, pH 6, followedby 3-fold dilution with the inclusion of Nonidet P-40 (1.17%, v/v, finalconcentration), sodium phosphate (200 mM, final concentration), and endoF (7 units/ml, final concentration). Conditions of endo H treatment weresimilar except that SDS concentration was 0.5% (w/v) and endo H was usedat a concentration of 1 μg/ml. The results with endo F were the same asthose with N-glycanase, whereas endo H had no effect on the purified SCFmaterial.

A number of conclusions can be drawn from the glyosidase experimentsdescribed above. The various treatments with N-glycanase [which removesboth complex and high-mannose N-linked carbohydrate (Tarentino et al.,Biochemistry 24, 4665-4671) (1985)], endo F [which acts similarly toN-glycanase (Elder and Alexander, Proc. Natl. Acad. Sci. USA 79,4540-4544 (1982)], endo H [which removes high-mannose and certain hybridtype N-linked carbohydrate (Tarentino et al., Methods Enzymol. 50C,574-580 (1978)], neuraminidase (which removes sialic acid residues), andO-glycanase [which removes certain O-linked carbohydrates (Lambin etal., Biochem. Soc. Trans. 12, 599-600 (1984)], suggest that: bothN-linked and O-linked carbohydrates are present; most of the N-linkedcarbohydrate is of the complex type; and sialic acid is present, with atleast some of it being part of the O-linked moieties. Some informationabout possible sites of N-linkage can be obtained from amino acidsequence data (Example 2). The fact that treatment with N-glycanase,endo F, and N-glycanase/neuraminidase can convert the heterogeneousmaterial apparent by SDS-PAGE to faster-migrating forms which are muchmore homogeneous is consistent with the conclusion that all of thematerial represents the same polypeptide, with the heterogeneity beingcaused by heterogeneity in glycosylation. It is also noteworthy that thesmallest forms obtained by the combined treatments with the variousglycosidases are in the range of M_(r) 18,000-20,000, relative to themolecular weight markers used in the SDS-PAGE.

Confirmation that the diffusely-migrating material around the M_(r)31,000 position on SDS-PAGE represents biologically active material allhaving the same basic polypeptide chain is given by the fact that aminoacid sequence data derived from material migrating in this region (e.g.,after electrophoretic transfer and cyanogen bromide treatment; Example2) matches that demonstrated for the isolated gene whose expression byrecombinant DNA means leads to biologically-active material (Example 4).

EXAMPLE 2 Amino Acid Sequence Analysis of Mammalian SCF

A. Reverse-phase High Performance Liquid Chromatography (HPLC) ofPurified Protein

Approximately 5 μg of SCF purified as in Example 1 (concentration=0.117mg/ml) was subjected to reverse-phase HPLC using a C₄ narrowbore column(Vydac, 300 Å widebore, 2 mm×15 cm). The protein was eluted with alinear gradient from 97% mobile phase A (0.1% trifluoroacetic acid)/3%mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) to 30%mobile phase A/70% mobile phase B in 70 min followed by isocraticelution for another 10 min at a flow rate of 0.2 ml per min. Aftersubtraction of a buffer blank chromatogram, the SCF was apparent as asingle symmetrical peak at a retention time of 70.05 min as shown inFIG. 10. No major contaminating protein peaks could be detected underthese conditions.

B. Sequencing of Electrophoretically-Transferred Protein Bands

SCF purified as in Example 1 (0.5-1.0 nmol) was treated as follows withN-glycanase, an enzyme which specifically cleaves the Asn-linkedcarbohydrate moieties covalently attached to proteins (see Example 1D).Six ml of the pooled material from fractions 4-6 of the C₄ column ofFIG. 5 was dried under vacuum. Then 150 μl of 14.25 mM CHAPS, 100 mM2-mercaptoethanol, 335 mM sodium phosphate, pH 8.6 was added andincubation carried out for 95 min at 37° C. Next 300 μl of 74 mM sodiumphosphate, 15 units/ml N-glycanase, pH 8.6 was added and incubationcontinued for 19 h. The sample was then run on a 9-18%SDS-polyacrylamide gradient gel (0.7 mm thickness, 20×20 cm). Proteinbands in the gel were electrophoretically transferred ontopolyvinyldifluoride (PVDF, Millipore Corp.) using 10 mM Caps buffer (pH10.5) at a constant current of 0.5 Amp for 1 h (Matsudaira, J. Biol.Chem., 261, 10035-10038 (1987)]. The transferred protein bands werevisualized by Coomassie Blue staining. Bands were present atM_(r ˜)29,000-33,000 and M_(r) ˜26,000, i.e., the deglycosylation wasonly partial (refer to Example 1D, FIG. 9); the former band representsundigested material and the latter represents material from whichN-linked carbohydrate is removed. The bands were cut out and directlyloaded (40% for M_(r) 29,000-33,000 protein and 80% for M_(r) 26,000protein) onto a protein sequencer (Applied Biosystems Inc., model 477).Protein sequence analysis was performed using programs supplied by themanufacturer [Hewick et al., J. Biol. Chem., 256 7990-7997 (1981)] andthe released phenylthiohydantoinyl amino acids were analyzed on-lineusing microbore C₁₈ reverse-phase HPLC. Both bands gave no signals for20-28 sequencing cycles, suggesting that both were unsequenceable bymethodology using Edman chemistry. The background level on eachsequencing run was between 1-7 pmol which was far below the proteinamount present in the bands. These data suggested that protein in thebands was N-terminally blocked.

C. In-situ CNBr Cleavage of Electrophoretically-Transferred Protein andSequencing

To confirm that the protein was in fact blocked, the membranes wereremoved from the sequencer (part B) and in situ cyanogen bromide (CNBr)cleavage of the blotted bands was carried out [CNBr (5%, w/v) in 70%formic acid for 1 h at 45° C.] followed by drying and sequence analysis.Strong sequence signals were detected, representing internal peptidesobtained from methionyl peptide bond cleavage by CNBr.

Both bands yielded identical mixed sequence signals listed below for thefirst five cycles.

Amino Acids Identified Cycle 1 Asp; Glu; Val; Ile; Leu Cycle 2 Asp; Thr;Glu; Ala; Pro; Val Cycle 3 Asn; Ser; His; Pro; Leu Cycle 4 Asp; Asn;Ala; Pro; Leu Cycle 5 Ser; Tyr; Pro

Both bands also yielded similar signals up to 20 cycles. The initialyields were 40-115 pmol for the M_(r) 26,000 band and 40-150 pmol forthe M_(r) 29,000-33,000 band. These values are comparable to theoriginal molar amounts of protein loaded onto the sequencer. The resultsconfirmed that protein bands corresponding to SCF contained a blockedN-terminus. Procedures used to obtain useful sequence information forN-terminally blocked proteins include: (a) deblocking the N-terminus(see section D); and (b) generating peptides by internal cleavages byCNBr (see Section E), by trypsin (see Section F), and by Staphylococcusaureus (strain V-8) protease (Glu-C) (see Section G). Sequence analysiscan proceed after the blocked N-terminal amino acid is removed or thepeptide fragments are isolated. Examples are described in detail below.

D. Sequence Analysis of BRL Stem Cell Factor Treated with PyroglutamicAcid Aminopeptidase

The chemical nature of the blockage moiety present at the amino terminusof SCF was difficult to predict. Blockage can be post-translational invivo [F. Wold, Ann. Rev. Biochem., 50, 783-814 (1981)] or may occur invitro during purification. Two post-translational modifications are mostcommonly observed. Acetylation of certain N-terminal amino acids such asAla, Ser, etc. can occur, catalyzed by N-α-acetyl transferase. This canbe confirmed by isolation and mass spectrometric analysis of anN-terminally blocked peptide. If the amino terminus of a protein isglutamine, deamidation of its gamma-amide can occur. Cyclizationinvolving the gamma-carboxylate and the free N-terminus can then occurto yield pyroglutamate. To detect pyroglutamate, the enzymepyroglutamate aminopeptidase can be used. This enzyme removes thepyroglutamate residue, leaving a free amino terminus starting at thesecond amino acid. Edman chemistry can then be used for sequencing.

SCF (purified as in Example 1; 400 pmol) in 50 mM sodium phosphatebuffer (pH 7.6 containing dithiothreitol and EDTA) was incubated with1.5 units of calf liver pyroglutamic acid aminopeptidase (pE-AP) for 16h at 37° C. After reaction the mixture was directly loaded onto theprotein sequencer. A major sequence could be identified through 46cycles. The initial yield was about 40% and repetitive yield was 94.2%.The N-terminal sequence of SCF including the N-terminal pyroglutamicacid is:

pE-AP cleavage site        ↓                                 10pyroGlu-Glu-Ile-Cys-Arg-Asn-Pro-Val-Thr-Asp-Asn-Val-Lys-Asp-Ile-Thr-Lys-(SEQ ID NO.:14)          20                                      30Leu-Val-Ala-Asn-Leu-Pro-Asn-Asp-Tyr-Met-Ile-Thr-Leu-Asn-Tyr-Val-                         40Ala-Gly-Met-Asp-Val-Leu-Pro-Ser-His-xxx-Trp-Leu-Arg-Asp-.........              xxx, not assigned at position 43

These results indicated that SCF contains pyroglutamic acid as itsN-terminus.

E. Isolation and Sequence Analysis of CNBr Peptides

SCF purified as in Example 1 (20-28 μg; 1.0-1.5 nmol) was treated withN-glycanase as described in Example 1. Conversion to the M_(r) 26,000material was complete in this case. The sample was dried and digestedwith CNBr in 70% formic acid (5%) for 18 h at room temperature. Thedigest was diluted with water, dried, and redissolved in 0.1%trifluoroacetic acid. CNBr peptides were separated by reverse-phase HPLCusing a C₄ narrowbore column and elution conditions identical to thosedescribed in Section A of this Example. Several major peptide fractionswere isolated and sequenced, and the results are summarized in thefollowing:

Retention Time Peptide (min) Sequence⁴ CB-4 15.5 L-P-P--- CB-6¹ 22.1 a.I-T-L-N-Y-V-A-G-(M) (SEQ ID NO.: 65) b.V-A-S-D-T-S-D-C-V-L-S-_-_-L-G-P-E-K-D- S-R-V-S-V-(_)-K---- (SEQ ID NO.:66) CB-8 28.0 D-V-L-P-S-H-C-W-L-R-D-(M) (SEQ ID NO.: 67) CB-10 30.1(containing sequence of CB-8) CB-15² 43.0E-E-N-A-P-K-N-V-K-E-S-L-K-K-P-T-R-(N)-F--T-P-E-E-F-F-S-I-F-D³-R-S-I-D-A------ (SEQ ID NO.: 68) CB-14 37.3 Bothpeptides contain identical sequence and to CB-15 CB-16 ¹Amino acids werenot detected at positions 12, 13 and 25. Peptide b was not sequenced tothe end. ²(N) in CB-15 was not detected; it was inferred based on thepotential N-linked glycosylation site. The peptide was not sequenced tothe end. ³Designates site where Asn may have been converted into Aspupon N-glycanase removal of N-linked sugar. ⁴Single letter code wasused: A,Ala; C,Cys; D,Asp; E,Glu; F,Phe; G,Gly; H,His; I,Ile; K,Lys;L,Leu; M,Met; N,Asn; P,Pro; Q,Gln; R,Arg; S,Ser; T,Thr; V,Val; W,Trp;and Y,Tyr.

F. Isolation and Sequencing of BRL Stem Cell Factor Tryptic Fragments

SCF purified as in Example 1 (20 μg in 150 μl 0.1 M ammoniumbicarbonate) was digested with 1 μg of trypsin at 37° C. for 3.5 h. Thedigest was immediately run on reverse-phase narrow bore C₄ HPLC usingelution conditions identical to those described in Section A of thisExample. All eluted peptide peaks had retention times different fromthat of undigested SCF (Section A). The sequence analyses of theisolated peptides are shown below:

Retention Time Peptide (min) Sequence⁴ T-1  7.1 E-S-L-K-K-P-E-T-R (SEQID NO.: 69) T-2¹ 28.1 V-S-V-(_)-K (SEQ ID NO.: 70) T-3 32.4I-V-D-D-L-V-A-A-M-E-E-N-A-P-K (SEQ ID NO.: 71) T-4² 40.0N-F-T-P-E-E-F-F-S-I-F-(_)-R (SEQ ID NO.: 72) T-5³ 46.4 a.L-V-A-N-L-P-N-D-Y-M-I-T-L-N-Y-V-A-G- M-D-V-L-P-S-H-C-W-L-R (SEQ ID NO.:73) b. S-I-D-A-F-K-D-F-M-V-A-S-D-T-S-D-C-V- L-S-(_)-(_)-L-G----(SEQ IDNO.: 74) T-7⁴ 72.8 E-S-L-K-K-P-E-T-R-(N)-F-T-P-E-E-F-F- S-I-F-(_)-R (SEQID NO.: 75) T-8 73.6 E-S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I- F-D-R (SEQID NO.: 76) ¹Amino acid at position 4 was not assigned. ²Amino acid atposition 12 was not assigned. ³Amino acids at positions 20 and 21 in 6of peptide T-5 were not identified; they were tentatively assigned asO-linked sugar attachment sites. ⁴Amino acid at position 10 was notdetected; it was inferred as Asn based on the potential N-linkedglycosylation site. Amino acid at position 21 was not detected.

G. Isolation and Sequencing of BRL Stem Cell Factor Peptides after S.aureus Glu-C Protease Cleavage

SCF purified as in Example 1 (20 μg in 150 μl 0.1 M ammoniumbicarbonate) was subjected to Glu-C protease cleavage at aprotease-to-substrate ratio of 1:20. The digestion was accomplished at37° C. for 18 h. The digest was immediately separated by reverse-phasenarrowbore C₄ HPLC. Five major peptide fractions were collected andsequenced as described below:

Retention Time Peptide (min) Sequence⁴ S-1  5.1 N-A-P-K-N-V-K-E (SEQ IDNO.: 77) S-2¹ 27.7 S-R-V-S-V-(_)-K-P-F-M-L-P-P-V-A-(A) (SEQ ID NO.: 78)S-3² 46.3 No sequence detected S-5³ 71.0S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I-F- (N)-R-S-I-D-A-F-K-D-F-M-V-A-S-D(SEQ ID NO.: 79) S-6³ 72.6 S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I-F-(N)-R-S-I-D-A-F-K-D-F-M-V-A-S-D-T-S-D (SEQ ID NO.: 80) ¹Amino acid atposition 6 of S-2 peptide was not assigned; this could be an O-linkedsugar attachment site. The Ala at position 16 of S-2 peptide wasdetected in low yield. ²Peptide S-3 could be the N-terminally blockedpeptide derived from the N-terminus of SCF. ³N in parentheses wasassigned as a potential N-linked sugar attachment site.

H. Sequence Analysis of BRL Stem Cell Factor after BNPS-skatole Cleavage

SCF (2 μg) in 10 mM ammonium bicarbonate was dried to completeness byvacuum centrifugation and then redissolved in 100 ul of glacial aceticacid. A 10-20 fold molar excess of BNPS-skatole was added to thesolution and the mixture was incubated at 50° C. for 60 min. Thereaction mixture was then dried by vacuum centrifugation. The driedresidue was extracted with 100 μl of water and again with 50 μl ofwater. The combined extracts were then subjected to sequence analysis asdescribed above. The following sequence was detected:

1                                    10Leu-Arg-Asp-Met-Val-Thr-His-Leu-Ser-Val-Ser-Leu-Thr-Thr-Leu-Leu- (SEQ IDNO.:81)              20                                        30Asp-Lys-Phe-Ser-Asn-Ile-Ser-Glu-Gly-Leu-Ser-(Asn)-Tyr-Ser-Ile-Ile-                             40 Asp-Lys-Leu-Gly-Lys-Ile-Val-Asp----

Position 28 was not positively assigned; it was assigned as Asn based onthe potential N-linked glycosylation site.

I. C-Terminal Amino Acid Determination of BRL Stem Cell Factor

An aliquot of SCF protein (500 pmol) was buffer-exchanged into 10 mMsodium acetate, pH 4.0 (final volume of 90 μl) and Brij-35 was added to0.05% (w/v). A 5 μl aliquot was taken for quantitation of protein. Fortyμl of the sample was diluted to 100 μl with the buffer described above.Carboxypeptidase P (from Penicillium janthinellum) was added at anenzyme-to-substrate ratio of 1:200. The digestion proceeded at 25° C.and 20 μl aliquots were taken at 0, 15, 30, 60 and 120 min. Thedigestion was terminated at each time point by adding trifluoroaceticacid to a final concentration of 5%. The samples were dried and thereleased amino acids were derivatized by reaction with Dabsyl chloride(dimethylaminoazobenzenesulfonyl chloride) in 0.2 M NaHCO₃ (pH 9.0) at70° C. for 12 min [Chang et al., Methods Enzymol., 90, 41-48 (1983)].The derivatized amino acids (one-sixth of each sample) were analyzed bynarrowbore reverse-phase HPLC with a modification of the procedure ofChang et al. [Techniques in Protein Chemistry, T. Hugli ed., Acad.Press, NY (1989), pp. 305-311]. Quantitative composition results at eachtime point were obtained by comparison to derivatized amino acidstandards (1 pmol). At 0 time, contaminating glycine was detected.Alanine was the only amino acid that increased with incubation time.After 2 h incubation, Ala was detected at a total amount of 25 pmol,equivalent to 0.66 mole of Ala released per mole of protein. This resultindicated that the natural mammalian SCF molecule contains Ala as itscarboxyl terminus, consistent with the sequence analysis of a C-terminalpeptide, S-2, which contains C-terminal Ala. This conclusion is alsoconsistent with the known specificity of carboxypeptidase P [Lu et al.,J. Chromatog. 447, 351-364 (1988)]. For example, cleavage ceases if thesequence Pro-Val is encountered. Peptide S-2 has the sequenceS-R-V-S-V-(T)-K-P-F-M-L-P-P-V-A-(A) (SEQ ID NO:82) and was deduced to bethe C-terminal peptide of SCF (see Section J in this Example). TheC-terminal sequence of - - - P-V-A-(A) (SEQ ID NO:83) restricts theprotease cleavage to alanine only. The amino acid composition of peptideS-2 indicates the presence of 1 Thr, 2 Ser, 3 Pro, 2 Ala, 3 Val, 1 Met,1 Leu, 1 Phe, 1 Lys, and 1 Arg, totalling 16 residues. The detection of2 Ala residues indicates that there may be two Ala residues at theC-terminus of this peptide (see table in Section G). Thus the BRL SCFterminates at Ala 164 or Ala 165.

J. Sequence of SCF

By combining the results obtained from sequence analysis of (1) intactstem cell factor after removing its N-terminal pyroglutamic acid, (2)the CNBr peptides, (3) the trypsin peptides, and (4) the Glu-C peptidasefragments, an N-terminal sequence and a C-terminal sequence were deduced(FIG. 11). The N-terminal sequence starts at pyroglutamic acid and endsat Met-48. The C-terminal sequence contains 84/85 amino acids (position82 to 164/165). The sequence from position 49 to 81 was not detected inany of the peptides isolated. However, a sequence was detected for alarge peptide after BNPS-skatole cleavage of BRL SCF as described inSection H of this Example. From these additional data, as well as DNAsequence obtained from rat SCF (Example 3) the N- and C-terminalsequences can be aligned and the overall sequence delineated as shown inFIG. 11. The N-terminus of the molecule is pyroglutamic acid and theC-terminus is alanine as confirmed by pyroglutamate aminopeptidasedigestion and carboxypeptidase P digestion, respectively.

From the sequence data, it is concluded that Asn-72 is glycosylated;Asn-109 and Asn-120 are probably glycosylated in some molecules but notin others. Asn-65 could be detected during sequence analysis andtherefore may only be partially glycosylated, if at all. Ser-142,Thr-143 and Thr-155, predicted from DNA sequence, could not be detectedduring amino acid sequence analysis and therefore could be sites ofO-linked carbohydrate attachment. These potential carbohydrateattachment sites are indicated in FIG. 11; N-linked carbohydrate isindicated by solid bold lettering; O-linked carbohydrate is indicated byopen bold lettering.

K. Amino Acid Compositional Analysis of BRL Stem Cell Factor

Material from the C₄ column of FIG. 7 was prepared for amino acidcomposition analysis by concentration and buffer exchange into 50 mMammonium bicarbonate.

Two 70 μl samples were separately hydrolyzed in 6 N HCl containing 0.1%phenol and 0.05% 2-mercaptoethanol at 110° C. in vacuo for 24 h. Thehydrolysates were dried, reconstituted into sodium citrate buffer, andanalyzed using ion exchange chromatography (Beckman Model 6300 aminoacid analyzer). The results are shown in Table 3. Using 164 amino acids(from the protein sequencing data) to calculate amino acid compositiongives a better match to predicted values than using 193 amino acids (asdeduced from PCR-derived DNA sequencing data, FIG. 14C).

TABLE 3 Quantitative Amino Acid Composition of Mammalian Derived SCFAmino Acid Composition Predicted Moles per mole of protein¹ Residues permolecule² Amino Acid Run #1 Run #2 (A) (B) Asx 24.46 24.26 25 28 Thr10.37 10.43 11 12 Ser 14.52 14.30 16 24 Glx 11.44 11.37 10 10 Pro 10.9010.85  9 10 Gly  5.81  6.20  4  5 Ala  8.62  8.35 7/8  8 Cys nd nd  4  5Val 14.03 13.96 15 15 Met  4.05  3.99  6  7 Ile  8.31  8.33  9 10 Leu17.02 16.97 16 19 Tyr  2.86  2.84  3  7 Phe  7.96  7.92  8  8 His  2.11 2.11  2  3 Lys 10.35 11.28 12 14 Trp nd nd  1  1 Arg  4.93  4.99  5  6Total 158    158    164/165 193  Calculated molecular weight 18,424³¹Based on 158 residues from protein sequence analysis (excluding Cys andTrp). ²Theoretical values calculated from protein sequence data (A) orfrom DNA sequence data (B). ³Based on 1-164 sequence.

Inclusion of a known amount of an internal standard in the amino acidcomposition analyses also allowed quantitation of protein in the sample;a value of 0.117 mg/ml was obtained for the sample analyzed.

EXAMPLE 3 Cloning of the Genes for Rat and Human SCF

A. Amplification and Sequencing of Rat SCF cDNA Fragments

Determination of the amino acid sequence of fragments of the rat SCFprotein made it possible to design mixed sequence oligonucleotidesspecific for rat SCF. The oligonucleotides were used as hybridizationprobes to screen rat cDNA and genomic libraries and as primers inattempts to amplify portions of the cDNA using polymerase chain reaction(PCR) strategies ([Mullis et al., Methods in Enzymol. 155, 335-350(1987)]. The oligodeoxynucleotides were synthesized by thephosphoramidite method [Beaucage, et al., Tetrahedron Lett., 22,1859-1862 (1981); McBride, et al., Tetrahedron Lett., 24, 245-248(1983)]; their sequences are depicted in FIG. 12A. The letters representA, adenine; T, thymine, C, cytosine;

G, guanine; I, inosine. The * in FIG. 12A represents oligonucleotideswhich contain restriction endonuclease recognition sequences. Thesequences are written 5′→3′.

A rat genomic library, a rat liver cDNA library, and two BRL cDNAlibraries were screened using ³²P-labelled mixed oligonucleotide probes,219-21 and 219-22 (FIG. 12A), whose sequences were based on amino acidsequence obtained as in Example 2. No SCF clones were isolated in theseexperiments using standard methods of cDNA cloning [Maniatis, et al.,Molecular Cloning, Cold Spring Harbor 212-246 (1982)].

An alternate approach which did result in the isolation of SCF nucleicacid sequences involved the use of PCR techniques. In this methodology,the region of DNA encompassed by two DNA primers is amplifiedselectively in vitro by multiple cycles of replication catalysed by asuitable DNA polymerase (such as TaqI DNA polymerase) in the presence ofdeoxynucleoside triphosphates in a thermo cycler. The specificity of PCRamplification is based on two oligonucleotide primers which flank theDNA segment to be amplified and hybridize to opposite strands. PCR withdouble-sided specificity for a particular DNA region in a complexmixture is accomplished by use of two primers with sequencessufficiently specific to that region. PCR with single-sided specificityutilizes one region-specific primer and a second primer which can primeat target sites present on many or all of the DNA molecules in aparticular mixture [Loh et al., Science, 243, 217-220 (1989)].

The DNA products of successful PCR amplification reactions are sourcesof DNA sequence information [Gyllensten, Biotechniques, 7, 700-708(1989)] and can be used to make labeled hybridization probes possessinggreater length and higher specificity than oligonucleotide probes. PCRproducts can also be designed, with appropriate primer sequences, to becloned into plasmid vectors which allow the expression of the encodedpeptide product.

The basic strategy for obtaining the DNA sequence of the rat SCF cDNA isoutlined in FIG. 13A. The small arrows indicate PCR amplifications andthe thick arrows indicate DNA sequencing reactions. PCRs 90.6 and 96.2,in conjunction with DNA sequencing, were used to obtain partial nucleicacid sequence for the rat SCF cDNA. The primers used in these PCRs weremixed oligonucleotides based on amino acid sequence depicted in FIG. 11.Using the sequence information obtained from PCRs 90.6 and 96.2, uniquesequence primers (224-27 and 224-28, FIG. 12A) were made and used insubsequent amplifications and sequencing reactions. DNA containing the5′ end of the cDNA was obtained in PCRs 90.3, 96.6, and 625.1 usingsingle-sided specificity PCR. Additional DNA sequence near theC-terminus of SCF protein was obtained in PCR 90.4. DNA sequence for theremainder of the coding region of rat SCF cDNA was obtained from PCRproducts 630.1, 630.2, 84.1 and 84.2 as described below in section C ofthis Example. The techniques used in obtaining the rat SCF cDNA aredescribed below.

RNA was prepared from BRL cells as described by Okayama et al. [MethodsEnzymol., 154, 3-28 (1987)]. PolyA+ RNA was isolated using an oligo(dT)cellulose column as described by Jacobson in [Methods in Enzymology,volume 152, 254-261 (1987)].

First-strand cDNA was synthesized using 1 μg of BRL polyA+ RNA astemplate and (dT)₁₂₋₁₈ as primer according to the protocol supplied withthe enzyme, Mo-MLV reverse transcriptase (Bethesda ResearchLaboratories). RNA strand degradation was performed using 0.14 M NaOH at84° C. for 10 min or incubation in a boiling water bath for 5 min.Excess ammonium acetate was added to neutralize the solution, and thecDNA was first extracted with phenol/chloroform, then extracted withchloroform/iso-amyl alcohol, and precipitated with ethanol. To makepossible the use of oligo(dC)-related primers in PCRs with single-sidedspecificity, a poly(dG) tail was added to the 3′ terminus of an aliquotof the first-strand cDNA with terminal transferase from calf thymus(Boeringer Mannheim) as previously described [Deng et al., MethodsEnzymol., 100, 96-103 (1983)].

Unless otherwise noted in the descriptions which follow, thedenaturation step in each PCR cycle was set at 94° C., 1 min; andelongation was at 72° C. for 3 or 4 min. The temperature and duration ofannealing was variable from PCR to PCR, often representing a compromisebased on the estimated requirements of several different PCRs beingcarried out simultaneously. When primer concentrations were reduced tolessen the accumulation of primer artifacts [Watson, Amplifications, 2,56 (1989)], longer annealing times were indicated; when PCR productconcentration was high, shorter annealing times and higher primerconcentrations were used to increase yield. A major factor indetermining the annealing temperature was the estimated T_(d) ofprimer-target association [Suggs et al., in Developmental Biology UsingPurified Genes eds. Brown, D. D. and Fox, C. F. (Academic, N.Y.) pp.683-693 (1981)]. The enzymes used in the amplifications were obtainedfrom either of three manufacturers: Stratagene, Promega, or Perkin-ElmerCetus. The reaction compounds were used as suggested by themanufacturer. The amplifications were performed in either a CoyTempcycle or a Perkin-Elmer Cetus DNA thermocycler.

Amplification of SCF cDNA fragments was usually assayed by agarose gelelectrophoresis in the presence of ethidium bromide and visualization byfluorescence of DNA bands stimulated by ultraviolet irradiation. In somecases where small fragments were anticipated, PCR products were analyzedby polyacrylamide gel electrophoresis. Confirmation that the observedbands represented SCF cDNA fragments was obtained by observation ofappropriate DNA bands upon subsequent amplification with one or moreinternally-nested primers. Final confirmation was by dideoxy sequencing[Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)] of thePCR product and comparison of the predicted translation products withSCF peptide sequence information.

In the initial PCR experiments, mixed oligonucleotides based on SCFprotein sequence were used [Gould, Proc. Natl. Acad. Sci. USA, 86,1934-1938 (1989)]. Below are descriptions of the PCR amplifications thatwere used to obtain DNA sequence information for the rat cDNA encodingamino acids −25 to 162.

In PCR 90.6, BRL cDNA was amplified with 4 pmol each of 222-11 and 223-6in a reaction volume of 20 μl. An aliquot of the product of PCR 90.6 waselectrophoresed on an agarose gel and a band of about the expected sizewas observed. One μl of the PCR 90.6 product was amplified further with20 pmol each of primers 222-11 and 223-6 in 50 μl for 15 cycles,annealing at 45° C. A portion of this product was then subjected to 25cycles of amplification in the presence of primers 222-11 and 219-25(PCR 96.2), yielding a single major product band upon agarose gelelectrophoresis. Asymmetric amplification of the product of PCR 96.2with the same two primers produced a template which was successfullysequenced. Further selective amplification of SCF sequences in theproduct of 96.2 was performed by PCR amplification of the product in thepresence of 222-11 and nested primer 219-21. The product of this PCR wasused as a template for asymmetric amplification and radiolabelled probeproduction (PCR2).

To isolate the 5′ end of the rat SCF cDNA, primers containing (dC)_(n)sequences, complimentary to the poly(dG) tails of the cDNA, wereutilized as non-specific primers. PCR 90.3 contained (dC)₁₂ (10 pmol)and 223-6 (4 pmol) as primers and BRL cDNA as template. The reactionproduct acted like a very high molecular weight aggregate, remainingclose to the loading well in agarose gel electrophoresis. One μl of theproduct solution was further amplified in the presence of 25 pmol of(dC)₁₂ and 10 pmol 223-6 in a volume of 25 ul for 15 cycles, annealingat 45° C. One-half μl of this product was then amplified for 25 cycleswith internally nested primer 219-25 and 201-7 (PCR 96.6). The sequenceof 201-7 is shown in FIG. 12C. No bands were observed by agarose gelelectrophoresis. Another 25 cycles of PCR, annealing at 40° C., wereperformed, after which one prominent band was observed. Southernblotting was carried out and a single prominent hybridizing band wasobserved. An additional 20 cycles of PCR (625.1), annealing at 45° C.,were performed using 201-7 and nested primer 224-27. Sequencing wasperformed after asymmetric amplification by PCR, yielding sequence whichextended past the putative amino terminus of the presumed signal peptidecoding sequence of pre-SCF. This sequence was used to designoligonucleotide primer 227-29 containing the 5′ end of the coding regionof the rat SCF cDNA. Similarly, the 3′ DNA sequence ending at amino acid162 was obtained by sequencing PCR 90.4 (see FIG. 13.A).

The sequence of the rat SCF coding region downstream of codon 162 wasobtained by direct sequencing of the products of PCRs in which rat SCF(+)-strand primers were combined with (−)-strand primers designed fromthe human SCF 3′-untranslated region sequence. Rat SCF primers 224-24(SEQ ID NO:10)(FIG. 12A) or 227-31 (5′-CCTGAGAAAGATTCCAGAGTC-3′) (SEQ IDNO:84). were used in combination with either of the two human SCFprimers 283-19 (5′-CTGCAGTTTGTATCTGAAG-3′) (SEQ ID NO: 85) or 283-20(5′-CATATAAAGTCATGGGTAG-3′) (SEQ ID NO:86). The rat SCF cDNA sequnce isshown in FIG. 14C.

B. Cloning of the Rat Stem Cell Factor Genomic DNA

Probes made from PCR amplification of cDNA encoding rat SCF as describedin section A above were used to screen a library containing rat genomicsequences (obtained from CLONTECH Laboratories, Inc.; catalog numberRL1022 j). The library was constructed in the bacteriophage λ vectorEMBL-3 SP6/T7 using DNA obtained from an adult male Sprague-Dawley rat.The library, as characterized by the supplier, contains 2.3×10⁶independent clones with an average insert size of 16 kb.

PCRs were used to generate ³²P-labeled probes used in screening thegenomic library. Probe PCR1 (FIG. 13A) was prepared in a reaction whichcontained 16.7 μM ³²P[alpha]-dATP, 200 μM dCTP, 200 μM dGTP, 200 μMdTTP, reaction buffer supplied by Perkin Elmer Cetus, Taq polymerase(Perkin Elmer Cetus) at 0.05 units/ml, 0.5 μM 219-26, 0.05 μM 223-6 and1 μl of template 90.1 containing the target sites for the two primers.Probe PCR 2 was made using similar reaction conditions except that theprimers and template were changed. Probe PCR 2 was made using 0.5 μM222-11, 0.05 μM 219-21 and 1 μl of a template derived from PCR 96.2.

Approximately 10⁶ bacteriophage were plated as described in Maniatis etal. [supra (1982)]. The plaques were transferred to GeneScreen Plus™filters (22 cm×22 cm; NEN/DuPont) which were denatured, neutralized anddried as described in a protocol from the manufacturer. Two filtertransfers were performed for each plate.

The filters were prehybridized in 1M NaCl, 1% SDS, 0.1% bovine serumalbumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone (hybridization solution)for approximately 16 h at 65° C. and stored at −20° C. The filters weretransfered to fresh hybridization solution containing ³²P-labeled PCR 1probe at 1.2×10⁵ cpm/ml and hybridized for 14 h at 65° C. The filterswere washed in 0.9 M NaCl, 0.09 M sodium citrate, 0.1% SDS, pH 7.2 (washsolution) for 2 h at room temperature followed by a second wash in freshwash solution for 30 min at 65° C. Bacteriophage clones from the areasof the plates corresponding to radioactive spots on autoradiograms wereremoved from the plates and rescreened with probes PCR1 and PCR2.

DNA from positive clones was digested with restriction endonucleasesBamHI, SphI or SstI, and the resulting fragments were subcloned intopUC119 and subsequently sequenced. The strategy for sequencing the ratgenomic SCF DNA is shown schematically in FIG. 14A. In this figure, theline drawing at the top represents the region of rat genomic DNAencoding SCF. The gaps in the line indicate regions that have not beensequenced. The large boxes represent exons for coding regions of the SCFgene with the corresponding encoded amino acids indicated above eachbox. The arrows represent the individual regions that were sequenced andused to assemble the consensus sequence for the rat SCF gene. Thesequence for rat SCF gene is shown in FIG. 14B.

Using PCR 1 probe to screen the rat genomic library, clonescorresponding to exons encoding amino acids 19 to 176 of SCF wereisolated. To obtain clones for exons upstream of the coding region foramino acid 19, the library was screened using oligonucleotide probe228-30. The same set of filters used previously with probe PCR 1 wereprehybridized as before and hybridized in hybridization solutioncontaining ³²P-labeled oligonucleotide 228-30 (0.03 picomole/ml) at 50°C. for 16 h. The filters were washed in wash solution at roomtemperature for 30 min followed by a second wash in fresh wash solutionat 45° C. for 15 min. Bacteriophage clones from the areas of the platescorresponding to radioactive spots on autoradiograms were removed fromthe plates and rescreened with probe 228-30. DNA from positive cloneswas digested with restriction endonucleases and subcloned as before.Using probe 228-30, clones corresponding to the exon encoding aminoacids −20 to 18 were obtained.

Several attempts were made to isolate clones corresponding to theexon(s) containing the 5′-untranslated region and the coding region foramino acids −25 to −21. No clones for this region of the rat SCF genehave been isolated.

C. Cloning Rat cDNA for Expression in Mammalian Cells

Mammalian cell expression systems were devised to ascertain whether anactive polypeptide product of rat SCF could be expressed in and secretedby mammalian cells. Expression systems were designed to expresstruncated versions of rat SCF (SCF¹⁻¹⁶² and SCF¹⁻¹⁶⁴) and a protein(SCF¹⁻¹⁹³) predicted from the translation of the gene sequence in FIG.14C.

The expression vector used in these studies was a shuttle vectorcontaining pUC119, SV40 and HTLVI sequences. The vector was designed toallow autonomous replication in both E. coli and mammalian cells and toexpress inserted exogenous DNA under the control of viral DNA sequences.This vector, designated V19.8, harbored in E. coli DH5, is depositedwith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. (ATCC# 68124). This vector is a derivative of pSVDM19described in Souza U.S. Pat. No. 4,810,643 hereby incorporated byreference.

The cDNA for rat SCF¹⁻¹⁶² was inserted into plasmid vector V19.8. ThecDNA sequence is shown in FIG. 14C. The cDNA that was used in thisconstruction was synthesized in PCR reactions 630.1 and 630.2, as shownin FIG. 13A. These PCRs represent independent amplifications andutilized synthetic oligonucleotide primers 227-29 and 227-30. Thesequence for these primers was obtained from PCR generated cDNA asdescribed in section A of this Example. The reactions, 50 μl in volume,consisted of 1× reaction buffer (from a Perkin Elmer Cetus kit), 250 μMdATP, 250 μM dCTP, 250 μM dGTP, and 250 μM dTTP, 200 ng oligo(dT)-primedcDNA, 1 picomole of 227-29, 1 picomole of 227-30, and 2.5 units of Taqpolymerase (Perkin Elmer Cetus). The cDNA was amplified for 10 cyclesusing a denaturation temperature of 94° C. for 1 min, an annealingtemperature of 37° C. for 2 min, and an elongation temperature of 72° C.for 1 min. After these initial rounds of PCR amplification, 10 picomolesof 227-29 and 10 picomoles of 227-30 were added to each reaction.Amplifications were continued for 30 cycles under the same conditionswith the exception that the annealing temperature was changed to 55° C.The products of the PCR were digested with restriction endonucleasesHindIII and SstII. V19.8 was similarly digested with HindIII and SstII,and in one instance, the digested plasmid vector was treated with calfintestinal alkaline phosphatase; in other instances, the large fragmentfrom the digestion was isolated from an agarose gel. The cDNA wasligated to V19.8 using T4 polynucleotide ligase. The ligation productswere transformed into competent E. coli strain DH5 as described[Okayama, et. al., supra (1987)]. DNA prepared from individual bacterialclones was sequenced by the Sanger dideoxy method. FIG. 17 shows aconstruct of V19.8 SCF. These plasmids were used to transfect mammaliancells as described in Example 4 and Example 5.

The expression vector for rat SCF¹⁻¹⁶⁴ was constructed using a strategysimilar to that used for SCF¹⁻¹⁶² in which cDNA was synthesized usingPCR amplification and subsequently inserted into V19.8. The cDNA used inthe constructions was synthesized in PCR amplifications with V19.8containing SCF¹⁻¹⁶² cDNA (V19.8:SCF¹⁻¹⁶²) as template, 227-29 as theprimer for the 5′-end of the gene and 237-19 as the primer for the3′-end of the gene. Duplicate reactions (50 ul) contained 1× reactionbuffer, 250 uM each of dATP, dCTP, dGTP and dTTP, 2.5 units of Taqpolymerase, 20 ng of V19.8:SCF¹⁻¹⁶², and 20 picomoles of each primer.The cDNA was amplified for 35 cycles using a denaturation temperature of94° C. for 1 min, an annealing temperature of 55° C. for 2 min and anelongation temperature of 72° C. for 2 min. The products of theamplifications were digested with restriction endonucleases HindIII andSstII and inserted into V19.8. The resulting vector contains the codingregion for amino acids −25 to 164 of SCF followed by a terminationcodon.

The cDNA for a 193 amino acid form of rat SCF, (rat SCF¹⁻¹⁹³ ispredicted from the translation of the DNA sequence in FIG. 14C) was alsoinserted into plasmid vector V19.8 using a protocol similar to that usedfor the rat SCF¹⁻¹⁶². The cDNA that was used in this construction wassynthesized in PCR reactions 84.1 and 84.2 (FIG. 13A) utilizingoligonucleotides 227-29 and 230-25. The two reactions representindependent amplifications starting from different RNA preparations. Thesequence for 227-29 was obtained via PCR reactions as described insection A of this Example and the sequence for primer 230-25 wasobtained from rat genomic DNA (FIG. 14B). The reactions, 50 μl involume, consisted of 1× reaction buffer (from a Perkin Elmer Cetus kit),250 μM dATP, 250 μM dCTP, 250 μM dGTP, and 250 μM dTTP, 200 ngoligo(dT)-primed cDNA, 10 picomoles of 227-29, 10 picomoles of 230-25,and 2.5 units of Taq polymerase (Perkin Elmer Cetus). The cDNA wasamplified for 5 cycles using a denaturation temperature of 94° C. for 1½minutes, an annealing temperature of 50° C. for 2 min, and an elongationtemperature of 72° C. for 2 min. After these initial rounds, theamplifications were continued for 35 cycles under the same conditionswith the exception that the annealing temperature was changed to 60° C.The products of the PCR amplification were digested with restrictionendonucleases HindIII and SstII. V19.8 DNA was digested with HindIII andSstII and the large fragment from the digestion was isolated from anagarose gel. The cDNA was ligated to V19.8 using T4 polynucleotideligase. The ligation products were transformed into competent E. colistrain DH5 and DNA prepared from individual bacterial clones wassequenced. These plasmids were used to transfect mammalian cells inExample 4.

D. Amplification and Sequencing of Human SCF cDNA PCR Products

The human SCF cDNA was obtained from a hepatoma cell line HepG2 (ATCC HB8065) using PCR amplification as outlined in FIG. 13B. The basicstrategy was to amplify human cDNA by PCR with primers whose sequencewas obtained from the rat SCF cDNA.

RNA was prepared as described by Maniatis et al. [supra (1982)]. PolyA+RNA was prepared using oligo dT cellulose following manufacturersdirections. (Collaborative Research Inc.).

First strand cDNA was prepared as described above for BRL cDNA, exceptthat synthesis was primed with 2 μM oligonucleotide 228-28, shown inFIG. 12C, which contains a short random sequence at the 3′ end attachedto a longer unique sequence. The unique-sequence portion of 228-28provides a target site for amplification by PCR with primer 228-29 asnon-specific primer. Human cDNA sequences related to at least part ofthe rat SCF sequence were amplified from the HepG2 cDNA by PCR usingprimers 227-29 and 228-29 (PCR 22.7, see FIG. 13B; 15 cycles annealingat 60° C. followed by 15 cycles annealing at 55° C). Agarose gelelectrophoresis revealed no distinct bands, only a smear of apparentlyheterogeneously sized DNA. Further preferential amplification ofsequences closely related to rat SCF cDNA was attempted by carrying outPCR with 1 μl of the PCR 22.7 product using internally nested rat SCFprimer 222-11 and primer 228-29 (PCR 24.3; 20 cycles annealing at 55°C.). Again only a heterogeneous smear of DNA product was observed onagarose gels. Double-sided specific amplification of the PCR 24.3products with primers 222-11 and 227-30 (PCR 25.10; 20 cycles) gave riseto a single major product band of the same size as the corresponding ratSCF cDNA PCR product. Sequencing of an asymmetric PCR product (PCR 33.1)DNA using 224-24 as sequencing primer yielded about 70 bases of humanSCF sequences.

Similarly, amplification of 1 μl of the PCR 22.7 product, first withprimers 224-25 and 228-29 (PCR 24.7, 20 cycles), then with primers224-25 and 227-30 (PCR 41.11) generated one major band of the same sizeas the corresponding rat SCF product, and after asymmetric amplification(PCR 42.3) yielded a sequence which was highly homologous to the rat SCFsequence when 224-24 was used as sequencing primer. Unique sequenceoligodeoxynucleotides targeted at the human SCF cDNA were synthesizedand their sequences are given in FIG. 12B.

To obtain the human counterpart of the rat SCF PCR-generated codingsequence which was used in expression and activity studies, a PCR withprimers 227-29 and 227-30 was performed on 1 μl of PCR 22.7 product in areaction volume of 50 μl (PCR 39.1). Amplification was performed in aCoy Tempcycler. Because the degree of mismatching between the human SCFcDNA and the rat SCF unique primer 227-30 was unknown, a low stringencyof annealing (37° C.) was used for the first three cycles; afterwardannealing was at 55° C. A prominent band of the same size (about 590 bp)as the rat homologue appeared, and was further amplified by dilution ofa small portion of PCR 39.1 product and PCR with the same primers (PCR41.1). Because more than one band was observed in the products of PCR41.1, further PCR with nested internal primers was performed in order todetermine at least a portion of its sequence before cloning. After 23cycles of PCR with primers 231-27 and 227-29 (PCR 51.2), a single,intense band was apparent. Asymmetric PCRs with primers 227-29 and231-27 and sequencing confirmed the presence of the human SCF cDNAsequences. Cloning of the PCR 41.1 SCF DNA into the expression vectorV19.8 was performed as already described for the rat SCF 1-162 PCRfragments in Section C above. DNA from individual bacterial clones wassequenced by the Sanger dideoxy method.

E. Cloning of the Human Stem Cell Factor Genomic DNA

A PCR7 probe made from PCR amplification of cDNA, see FIG. 13B, was usedto screen a library containing human genomic sequences. A riboprobecomplementary to a portion of human SCF cDNA, see below, was used tore-screen positive plaques. PCR 7 probe was prepared starting with theproduct of PCR 41.1 (see FIG. 13B). The product of PCR 41.1 was furtheramplified with primers 227-29 and 227-30. The resulting 590 bp fragmentwas eluted from an agarose gel and reamplified with the same primers(PCR 58.1). The product of PCR 58.1 was diluted 1000-fold in a 50 μlreaction containing 10 pmoles 233-13 and amplified for 10 cycles. Afterthe addition of 10 pmoles of 227-30 to the reaction, the PCR wascontinued for 20 cycles. An additional 80 pmoles of 233-13 was added andthe reaction volume increased to 90 μl and the PCR was continued for 15cycles. The reaction products were diluted 200-fold in a 50 μl reaction,20 pmoles of 231-27 and 20 pmoles of 233-13 were added, and PCR wasperformed for 35 cycles using an annealing temperature of 48° inreaction 96.1. To produce ³²P-labeled PCR7, reaction conditions similarto those used to make PCR1 were used with the following exceptions: in areaction volume of 50 μl, PCR 96.1 was diluted 100-fold; 5 pmoles of231-27 was used as the sole primer; and 45 cycles of PCR were performedwith denaturation at 94° for 1 minute, annealing at 48° for 2 minutesand elongation at 72° for 2 minutes.

The riboprobe, riboprobe 1, was a ³²P-labelled single-stranded RNAcomplementary to nucleotides 2-436 of the hSCF DNA sequence shown inFIG. 15B. To construct the vector for the production of this probe, PCR41.1 (FIG. 13B) product DNA was digested with HindIII and EcoRI andcloned into the polylinker of the plasmid vector pGEM3 (Promega,Madison, Wis.). The recombinant pGEM3:hSCF plasmid DNA was thenlinearized by digestion with HindIII. ³²P-labeled riboprobe 1 wasprepared from the linearized plasmid DNA by runoff transcription with T7RNA polymerase according to the instructions provided by Promega. Thereaction (3 μl) contained 250 ng of linearized plasmid DNA and 20 μM³²P-rCTP (catalog #NEG-008H, New England Nuclear (NEN) with noadditional unlabeled CTP.

The human genomic library was obtained from Stratagene (La Jolla,Calif.; catalog #:946203). The library was constructed in thebacteriophage Lambda Fix II vector using DNA prepared from a Caucasianmale placenta. The library, as characterized by the supplier, contained2×10⁶ primary plaques with an average insert size greater than 15 kb.Approximately 10⁶ bacteriophage were plated as described in Maniatis, etal. [supra (1982)]. The plaques were transferred to Gene Screen Plus™filters (22 cm²; NEN/DuPont) according to the protocol from themanufacturer. Two filter transfers were performed for each plate.

The filters were prehybridized in 6×SSC (0.9 M NaCl, 0.09 M sodiumcitrate pH 7.5), 1% SDS at 60° C. The filters were hybridized in fresh6×SSC, 1% SDS solution containing ³²P-labeled PCR 7 probe at 2×10⁵cpm/ml and hybridized for 20 h at 62° C. The filters were washed in6×SSC, 1% SDS for 16 h at 62° C. A bacteriophage plug was removed froman area of a plate which corresponded to radioactive spots onautoradiograms and rescreened with probe PCR 7 and riboprobe 1. Therescreen with PCR 7 probe was performed using conditions similar tothose used in the initial screen. The rescreen with riboprobe 1 wasperformed as follows: the filters were prehybridized in 6×SSC, 1% SDSand hybridized at 62° C. for 18 h in 0.25 M NaPO₄, (pH 7.5), 0.25 MNaCl, 0.001 M EDTA, 15% formamide, 7% SDS and riboprobe at 1×10⁶ cpm/ml.The filters were washed in 6×SSC, 1% SDS for 30 min at 62° C. followedby 1×SSC, 1% SDS for 30 min at 62° C. DNA from positive clones wasdigested with restriction endonucleases Bam HI, SphI or SstI and theresulting fragments were subcloned into pUC119 and subsequentlysequenced.

Using probe PCR 7, a clone was obtained that included exons encodingamino acids 40 to 176 and this clone is deposited at the ATCC (deposit#40681). To obtain clones for additional SCF exons, the human genomiclibrary was screened with riboprobe 2 and oligonucleotide probe 235-29.The library was screened in a manner similar to that done previouslywith the following exceptions: the hybridization with probe 235-29 wasdone at 37° C. and the washes for this hybridization were for 1 h at 37°C. and 1 h at 44° C. Positive clones were rescreened with riboprobe 2,riboprobe 3 and oligonucleotide probes 235-29 and 236-31. Riboprobes 2and 3 were made using a protocol similar to that used to produceriboprobe 1, with the following exceptions: (a) the recombinantpGEM3:hSCP plasmid DNA was linearized with restriction endonucleasePvuII (riboprobe 2) or PstI (riboprobe 3) and (b) the SP6 RNA polymerase(Promega) was used to synthesize riboprobe 3.

FIG. 15A shows the strategy used to sequence human genomic DNA. In thisfigure, the line drawing at the top represents the region of humangenomic DNA encoding SCF. The gaps in the line indicate regions thathave not been sequenced. The large boxes represent exons for codingregions of the SCF gene with the corresponding encoded amino acidsindicated above each box. The sequence of the human SCF gene is shown inFIG. 15B. The sequence of human SCF cDNA obtained PCR techniques isshown in FIG. 15C.

The sequence of exons 7, 8 and 9, which include the coding region foramino acids 177 to 248, were obtained from a bacteriophage lambda cloneisolated as described above using PCR7 as probe.

To isolate a clone of exon 1 of the human SCF gene, a second genomiclibrary was screened. The library, purchased from Clontech (Palo Alto,Calif.; catalog #HL 1067 J), was constructed in bacteriophage lambdavector EMBL3 SP6/T7 and contained 2.5×10⁶ independent clones with anaverage insert size of 15 kb. Approximately 10⁶ clones were plated andscreened as described above using oligonucleotide probe 249-31(5′-ACTTGTGTCTTCTTCATAAGGAAAGGC-3) (SEQ ID NO:87)). A SacI restrictionfragment of the lambda clone was cloned into plasmid vector pGEM4 forsubsequent sequence analysis. The sequence of the human SCF geneincluding exons 1, 7, 8 and 9 is shown in FIG. 15D.

F. Sequence of the Human SCF cDNA 5′ Region

Sequencing of products from PCRs primed by two gene-specific primersreveals the sequence of the region bounded by the 3′ ends of the twoprimers. One-sided PCRs, as indicated in Example 3A, can yield thesequence of flanking regions. One-sided PCR was used to extend thesequence of the 5′-untranslated region of human SCF cDNA.

First strand cDNA was prepared from poly A+ RNA from the human bladdercarcinoma cell line 5637 (ATCC HTB 9) using oligonucleotide 228-28 (FIG.12C) as primer, as described in Example 3D. Tailing of this cDNA with dGresidues, followed by one-sided PCR amplification using primerscontaining (dC)n sequences in combination with SCF-specific primers,failed to yield cDNA fragments extending upstream (5′) of the knownsequence.

A small amount of sequence information was obtained from PCRamplification of products of second strand synthesis primed byoligonucleotide 228-28. The untailed 5637 first strand cDNA describedabove (about 50 ng) and 2 pmol of 228-28 were incubated with Klenowpolymerase and 0.5 mM each of dATP, dCTP, dGTP and dTTP at 10-12° C. for30 minutes in 10 uL of 1×Nick-translation buffer [Maniatis et al.,Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory(1982)]. Amplification of the resulting cDNA by sequential one-sidedPCRs with primer 228-29 in combination with nested SCF primers (in orderof use: 235-30, 233-14, 236-31 and finally 235-29) yielded complexproduct mixtures which appeared as smears on agarose gels. Significantenrichment of SCF-related cDNA fragments was indicated by the increasingintensity of the specific product band observed when comparable volumesof the successive one-sided PCR products were amplified with two SCFprimers (227-29 and 235-29, for example, yielding a product of about 150bp). Attempts to select for a particular size range of products bypunching out portions of the agarose gel smears and reamplifying by PCRin most cases failed to yield a well-defined band which containedSCF-related sequences.

One reaction, PCR 16.17, which contained only the 235-29 primer, gaverise to a band which apparently arose from priming by 235-29 at anunknown site 5′ of the coding region in addition to the expected site,as shown by mapping with the restriction enzymes PvuII and PstI and PCRanalysis with nested primers. This product was gel-purified andreamplified with primer 235-29, and sequencing was attempted by theSanger dideoxy method using ³²P-labelled primer 228-30. The resultingsequence was the basis for the design of oligonucleotide 254-9 (FIG.12B). When this 3′ directed primer was used in subsequent PCRs incombination with 5′ directed SCF primers, bands of the expected sizewere obtained. Direct Sanger sequencing of such PCR products yieldednucleotides 180 through 204 of a human SCF cDNA sequence, FIG. 15C.

In order to obtain more sequence at the 5′ end of the hSCF cDNA, firststrand cDNA was prepared from 5637 poly A⁺ RNA (about 300 ng) using anSCF-specific primer (2 pmol of 233-14) in a 16 uL reaction containing0.2 U MMLV reverse transcriptase (purchased from BRL) and 500 uM eachdNTP. After standard phenol-chloroform and chloroform extractions andethanol precipitation (from 1 M ammonium acetate) steps, the nucleicacids were resuspended in 20 uL of water, placed in a boiling water bathfor 5 minutes, then cooled and tailed with terminal transferase in thepresence of 8 uM dATP in a CoCl₂-containing buffer [Deng and Wu, Methodsin Enzymology, 100, pp. 96-103]. The product, (dA)_(n)-tailedfirst-strand cDNA was purified by phenol-chloroform extraction andethanol precipitation and resuspended in 20 uL of 10 mM tris, pH 8.0,and 1 mM EDTA.

Enrichment and amplification of human SCF-related cDNA 5′ end fragmentsfrom about 20 ng of the (dA)_(n)-tailed 5637 cDNA was performed asfollows: an initial 26 cycles of one-sided PCR were performed in thepresence of SCF-specific primer 236-31 and a primer or primer mixturecontaining (dT)_(n) sequences at or near the 3′ end, for instance primer221-12 or a mixture of primers 220-3, 220-7, and 220-11 (FIG. 12C). Theproducts (1 μl) of these PCRs were then amplified in a second set ofPCRs containing primers 221-12 and 235-29. A major product band ofapproximately 370 bp was observed in each case upon agarose gelanalysis. A gel plug containing part of this band was punched out of thegel with the tip of a Pasteur pipette and transferred to a smallmicrofuge tube. 10 uL of water was added and the plug was melted in an84° C. heating block. A PCR containing primers 221-12 and 235-29 (8 pmoleach) in 40 uL was inoculated with 2 uL of the melted, diluted gel plug.After 15 cycles, a slightly diffuse band of approximately 370 bp wasvisible upon agarose gel analysis. Asymmetric PCRs were performed togenerate top and bottom strand sequencing templates: for each reaction,4 uL of PCR reaction product and 40 pmol of either primer 221-12 orprimer 235-29 in a total reaction volume of 100 uL were subjected to 25cycles of PCR (1 minute, 95° C.; 30 seconds, 55° C.; 40 seconds, 72°C.). Direct sequencing of the 221-12 primed PCR product mixtures (afterthe standard extractions and ethanol precipitation) with ³²P-labelledprimer 262-13 (FIG. 12B) yielded the 5′ sequence from nucleotide 1 to179 (FIG. 15C).

G. Amplification and Sequencing of Human Genomic DNA at the Site of theFirst Coding Exon of the Stem Cell Factor

Screening of a human genomic library with SCF oligonucleotide probesfailed to reveal any clones containing the known portion of the firstcoding exon. An attempt was then initiated to use a one-sided PCRtechnique to amplify and clone genomic sequences surrounding this exon.

Primer extension of heat-denatured human placental DNA (purchased fromSigma) was performed with DNA polymerase I (Klenow enzyme, largefragment; Boehringer-Mannheim) using a non-SCF primer such as 228-28 or221-11 under non-stringent (low temperature) conditions, such as 12° C.,to favor priming at a very large number of different sites. Eachreaction was then diluted five-fold into TaqI DNA polymerase buffercontaining TaqI polymerase and 100 uM of each dNTP, and elongation ofDNA strands was allowed to proceed at 72° C. for 10 minutes. The productwas then enriched for stem cell factor first exon sequences by PCR inthe presence of an SCF first exon oligonucleotide (such as 254-9) andthe appropriate non-SCF primer (228-29 or 221-11). Agarose gelelectrophoresis revealed that most of the products were short (less than300 bp). To enrich for longer species, the portion of each agarose gellane corresponding to length greater than 300 bp was cut out andelectrophoretically eluted. After ethanol precipitation and resuspensionin water, the gel purified PCR products were cloned into a derivative ofpGEM4 containing an SfiI site as a HindIII to SfiI fragment.

Colonies were screened with a ³²P-labelled SCF first exonoligonucleotide. Several positive colonies were identified and thesequences of the inserts were obtained by the Sanger method. Theresulting sequence, which extends downstream from the first exon througha consensus exon-intron boundary into the neighboring intron, is shownin FIG. 15B.

H. Amplification and Sequencing of SCF cDNA Coding Regions from Mouse,Monkey, Dog, Cat, Cow and Chicken

First strand cDNA was prepared from total RNA or poly A⁺ RNA from monkeyliver (purchased from Clontech) and from the cell lines NIH-3T3 (mouse,ATCC CRL 1658), D17 (dog, ATCC CCL 183), bovine endothelial cell line(provided by Yves DeClerck, Childrens Hospital Los Angeles, Los Angeles,Calif.), feline embryonic fibroblast cell line (Jarrett et al., J. Gen.Virology, 20:169-175 (1973)) and chicken brain RNA. The primer used infirst strand cDNA synthesis was either the nonspecific primer 228-28 oran SCF primer (227-30, 237-19, 237-20, 230-25 or 241-6).

PCR amplification with primer 227-29 and one of the primers 227-30,237-19 or 237-20 in each case except chicken yielded a fragment of theexpected size which was sequenced either directly or after cloning intoV19.8 or a pGEM vector. Additional sequences near the 5′ end of the SCFcDNAs were obtained from PCR amplifications utilizing an SCF-specificprimer in combination with either 254-9 or one of the non-specificprimers 228-29 and 221-11. Additional sequences at the 3′ end of the SCFcoding regions were obtained after PCR amplification of 228-28 primedcDNA with combinations of SCF coding region (+)-strand primers with(−)-primers based on the human SCF 3′ untranslated region as describedin Example 3A. The primers 283-19 (SEQ ID NO:85) and 283-20 (SEQ IDNO:86) (Example 3A) and primer 287-9 (5′-TGTACGAAAGTAACAGTGTTG-3′) (SEQID NO:88) were used. In the case of chicken, amplification wasaccomplished with primers to 227-29 (SEQ ID NO:74) or 247-1(5′-ACTGCTCCTATTTAATCCTCTC-3′) (SEQ ID NO:89)in combination with 247-2(5′-CACTGACTCTGGAATCTTTCTCA-3′) (SEQ ID NO:90) or 287-9. The alignedamino acid sequences of human (FIG. 42), monkey, dog, mouse, rat, cat,cwo and chicken. SCF mature proteins are shown in FIG. 16.

The known SCF amino acid sequences are highly homologous throughout muchof their length. Identical consensus signal peptide sequences arepresent in the coding regions of all seven species. The amino acidexpected to be at the amino terminus of the mature protein by analogywith the rat SCF is designated by the numeral 1 in this figure. The dogand cow cDNA sequence contains an ambiguity which results in avaline/leucine ambiguity in the amino acid sequence at codon 129. Thehuman, monkey, rat and mouse amino acid sequences co-align without anyinsertions or deletions. The dog sequence has a single extra residue atposition 130 as compared to the other species. Human and monkey differat only one position, a conservative replacement of valine (human) byalanine (monkey) at position 130. The predicted SCF sequence immediatelybefore and after the putative processing site near residue 164 is highlyconserved between species.

EXAMPLE 4 Expression of Recombinant Rat SCF in COS-1 Cells

For transient expression in COS-1 cells (ATCC CRL 1650), vector V19.8(Example 3C) containing the rat SCF¹⁻¹⁶² and SCF¹⁻¹⁹³ genes wastransfected into duplicate 60 mm plates [Wigler et al., Cell, 14,725-731 (1978)]. The plasmid V19.8 SCF is shown in FIG. 17. As acontrol, the vector without insert was also transfected. Tissue culturesupernatants were harvested at various time points post-transfection andassayed for biological activity. Table 4 summarizes the HPP-CFC bioassayresults and Table 5 summarizes the MC/9 ³H-thymidine uptake data fromtypical transfection experiments. Bioassay results of supernatants fromCOS-1 cells transfected with the following plasmids are shown in Tables4 and 5: a C-terminally-truncated form of rat SCF with the C-terminus atamino acid position 162 (V19.8 rat SCF¹⁻¹⁶²), SCF¹⁻¹⁶² containing aglutamic acid at position 81 [V19.8 rat SCF¹⁻¹⁶² (Glu81)], and SCF¹⁻¹⁶²containing an alanine at position 19 [V19.8 rat SCF¹⁻¹⁶² (Ala19)]. Theamino acid substitutions were the product of PCR reactions performed inthe amplification of rat SCF¹⁻¹⁶² as indicated in Example 3. Individualclones of V19.8 rat SCF¹⁻¹⁶² were sequenced and two clones were found tohave amino acid substitutions. As can be seen in Tables 4 and 5, therecombinant rat SCF (also referred to throughout this application asrrat SCF or rrSCF), is active in the bioassays used to purify naturalmammalian SCF in Example 1.

TABLE 4 HPP-CFC Assay of COS-1 Supernatants from Cells Transfected withRat SCF DNA Volume of Colony Sample CM Assayed (μl) #/200,000 cellsV19.8 (no insert) 100  0 50 0 25 0 12 0 V19.8 rat SCF¹⁻¹⁶² 100  >50    50 >50     25 >50     12 >50      6 30   3 8 V19.8 rat SCF¹⁻¹⁶² 100  26 (Glu81) 50 10  25 2 12 0 V19.8 rat SCF¹⁻¹⁶² 100  41  (Ala19) 50 18  25 512 0  6 0  3 0

TABLE 5 MC/9³H-Thymidine Uptake Assay of COS-1 Supernatants from CellsTransfected with Rat SCF DNA Sample Volume of CM Assayed (μl) cpm V19.8(no insert) 25 1,936 12 2,252  6 2,182  3 1,682 V19.8 rat SCF¹⁻¹⁶² 2511,648  12 11,322   6 11,482   3 9,638 V19.8 rat SCF¹⁻¹⁶² 25 6,220(Glu81) 12 5,384  6 3,692  3 1,980 V19.8 rat SCF¹⁻¹⁶² 25 8,396 (Ala19)12 6,646  6 4,566  3 3,182

Recombinant rat SCF, and other factors, were tested individually in ahuman CFU-GM [Broxmeyer et al., supra (1977)] assay which measures theproliferation of normal bone marrow cells and the data are shown inTable 6. Results for COS-1 supernatants from cultures 4 days aftertransfection with V19.8 SCF¹⁻¹⁶² in combination with other factors arealso shown in Table 6. Colony numbers are the average of triplicatecultures.

The recombinant rat SCF has primarily a synergistic activity on normalhuman bone marrow in the CFU-GM assay. In the experiment in Table 6, SCFsynergized with human GM-CSF, human IL-3, and human CSF-1. In otherassays, synergy was observed with G-CSF also. There was someproliferation of human bone marrow after 14 days with rat SCF; however,the clusters were composed of <40 cells. Similar results were obtainedwith natural mammalian-derived SCF.

TABLE 6 Human CFU-GM Assay of COS-1 Supernatants from Cells Transfectedwith Rat SCF DNA Sample Colony #/100,000 cells (± SEM) Saline 0 GM-CSF 7 ± 1 G-CSF 24 ± 1 IL-3  5 ± 1 CSF-1 0 SCF¹⁻¹⁶² 0 GM-CSF + SCF¹⁻¹⁶² 29± 6 G-CSF + SCF¹⁻¹⁶² 20 ± 1 IL-3 + SCF¹⁻¹⁶² 11 ± 1 CSF-1 + SCF¹⁻¹⁶²  4 ±0

EXAMPLE 5 Expression of Recombinant SCF in Chinese Hamster Ovary Cells

This example relates to a stable mammalian expression system forsecretion of SCF from CHO cells (ATCC CCL 61 selected for DHFR-).

A. Recombinant Rat SCF

The expression vector used for SCF production was V19.8 (FIG. 17). Theselectable marker used to establish stable transformants was the genefor dihydrofolate reductase in the plasmid pDSVE.1. Plasmid pDSVE.1(FIG. 18) is a derivative of pDSVE constructed by digestion of PDSVE bythe restriction enzyme SalI and ligation to an oligonucleotide fragmentconsisting of the two oligonucleotides

5′TCGAC CCGGA TCCCC 3′ (SEQ ID NO:91)

3′ G GGCCT AGGGG AGCT 5′ (SEQ ID NO:92).

Vector pDSVE is described in commonly owned U.S. Ser. Nos. 025,344 and152,045 hereby incorporated by reference. The vector portion of V19.8and pDSVE.1 contain long stretches of homology including a bacterialColEl origin of replication and ampicillin resistance gene and the SV40origin of replication. This overlap may contribute to homologousrecombination during the transformation process, thereby facilitatingco-transformation.

Calcium phosphate co-precipitates of V19.8 SCF constructs and pDSVE.1were made in the presence or absence of 10 μg of carrier mouse DNA using1.0 or 0.1 μg of pDSVE.1 which had been linearized with the restrictionendonuclease PvuI and 10 μg of V19.8 SCF as described [Wigler et al.,supra (1978)]. Colonies were selected based upon expression of the DHFRgene from pDSVE.1. Colonies capable of growth in the absence of addedhypoxanthine and thymidine were picked using cloning cylinders andexpanded as independent cell lines. Cell supernatants from individualcell lines were tested in an MC/9 ³H-thymidine uptake assay. Resultsfrom a typical experiment are presented in Table 7.

TABLE 7 MC/9 ³H-Thymidine Uptake Assay of Stable CHO Cell SupernatantsFrom Cells Transfected With Rat SCF DNA Volume of ConditionedTransfected DNA Medium Assayed cpm V19.8 SCF¹⁻¹⁶² 25  33,926 12  34,9736 30,657 3 14,714   1.5  7,160 None 25    694 12   1,082 6   880 3   6721  1,354

B. Recombinant Human SCF

Expression of SCF in CHO cells was also achieved using the expressionvector pDSVRα2 which is described in commonly owned Ser. No. 501,904filed Mar. 29, 1990, hereby incorporated by reference. This vectorincludes a gene for the selection and amplification of clones based onexpression of the DHFR gene. The clone pDSRα2 SCF was generated by a twostep process. The V19.8 SCF was digested with the restriction enzymeBamHI and the SCF insert was ligated into the BamHI site of pGEM3. DNAfrom pGEM3 SCF was digested with HindIII and SalI and ligated intopDSRα2 digested with HindIII and SalI. The same process was repeated forhuman genes encoding a COOH-terminus at the amino acid positions 162,164 and 183 of the sequence shown in FIG. 15C.

Genes encoding proteins with the COOH-terminus at position 248 of thesequences shown in FIG. 42 and amino acids 1-220 of the sequence in FIG.44 were generated as follows: DNA encoding the 1-164 amino acid SCFinsert in pGEM3 was isolated by digestion with HindIII and ligated intothe HindIII site of M13mp18. The sequence preceding the ATG initiationcodon was changed by site directed mutagenesis using the oligonucleotide

5′-TCTTCTTCATGGCGGCGGCAAGCTT-3′ (SEQ ID NO:93)

and a kit from Amersham (Arlington Heights, Ill.). The resulting clonewas digested with HindIII and the SCF sequences were ligated to pDSRα2digested with HindIII. This clone was designated pDSRα2-Δ12. The 3′ endof this gene was exchanged with the 3′ end of the 248 or 220 sequencesby digesting pDSRα2-Δ12 with XbaI, filling in the resulting ends withDNA polymerase I (Klenow fragment) and dATP, dCTP, dGTP and TTP togenerate a blunt end and subsequent digestion with SpeI. The 220 and 248sequences were digested with DraI, which leaves a blunt end and SpeI.The vector and inserts were then ligated together to generate pDSRα2-Δ23(248 amino acid sequence) or pDSRα2-Δ220 (220 amino acid sequence).These plasmids were used to generate cell lines by calcium phosphateprecipitation as described in Example 5A except that pDSVE.1 was notused for selection.

Established cell lines were challenged with methotrexate [Shimke, inMethods in Enzymology, 151 85-104 (1987)] at 10 nM to increaseexpression levels of the DHFR gene and the adjacent SCF gene. Expressionlevels of recombinant human SCF were assayed by radioimmune assay, as inExample 7, and/or induction of colony formation in vitro using humanperipheral blood leucocytes. This assay is performed as described inExample 9 (Table 12) except that peripheral blood is used instead ofbone marrow and the incubation is performed at 20% O₂, 5% CO₂, and 75%N₂ in the presence of human EPO (10 U/ml). Results from typicalexperiments are shown in Table 8. The SCF²²⁰ and SCF²⁴⁸ also showedsimilar expression in these assays and as determined by Western blotanalysis. The CHO clone expressing human SCF¹⁻¹⁶⁴ has been deposited onSep. 25, 1990 with ATCC (CRL 10557) and designated HU164SCF17.

TABLE 8 hPBL Colony Assay of Conditioned Media From Stable CHO CellLines Transfected With Human SCF DNA Media Number of Transfected DNAassayed (μl) Colonies/10⁵ pDSRα2 hSCF¹⁻¹⁶⁴ 50 53 25 45 12.5 27 6.25 13pDSRα2 hSCF¹⁻¹⁶² 10 43 5 44 2.5 31 1.25 17 0.625 21 None (CHO control)50  4

C. Secreted Product of CHO Cells Transfected with pDSRα2-Δ23.

CHO cells transfected with pDSRα2-Δ23 (248 amino acid sequence; seeExample 5B) were cultured as described in Example 11A. As previouslydescribed, the sequences shown in FIG. 42 include a putative hydrophobictransmembrane region represented by amino acids numbered 190-212, whichcould anchor a synthesized protein in the cell membrane. This is alsothe case for the encoded rat sequences of FIG. 14, yet soluble rat SCFrepresenting amino acids 1-164/165 was recovered from conditioned mediumof BRL-3A cells as described in Examples 1 and 2. This is indicative ofproteolytic processing leading to release of soluble SCF. To study suchprocessing for a case involving the human protein, the CHO cellstransfected with pDSRα2-Δ23 were cultured as described in Example 5B.Conditioned medium contained soluble human SCF, which was purifiedessentially by the methods outlined in Example 11B. By SDS-PAGE,combined with the use of glycosidases as outlined in Examples 10 and11C, it was found that the behavior of the purified material was muchlike that described for BRL-3A derived rat SCF (Example 1D) and forhuman SCF purified from conditioned medium of CHO cells transfected withpDSRα2 human SCF¹⁻¹⁶² (see Example 11C). The mobility on SDS-PAGE of themajor band remaining after treatment with neuraminidase, O-glycanase,and N-glycanase was slightly less that the mobility seen for the majorband after such treatment of the CHO cell-derived human SCF 1-162described in Example 11C. This mobility difference corresponded to lessthan 1000 in molecular weight difference and indicated that the lessmobile product was larger by a few amino acids.

The purified material from the CHO cells transfected with pDSRα2-Δ23 wassubjected to detailed structural analysis, by methods including thosegiven in Example 2. The N-terminal amino acid sequence is Glu-Gly-Ile .. . , indicating that it is the product of processing/cleavage betweenresidues indicated as numbers (−1) Thr and (+1) (Glu) in FIG. 42.

To determine the precise C-terminal processing site(s), the purifiedmaterial was subjected to AspN peptidase digestion (20-50 μg SCF in100-200 μl 0.1 M sodium phosphate, pH 7.2, for 18 h at 37° C. withAspN:SCF ratio of 1:200 by weight) followed by HPLC to isolate resultingpeptides. The elution profile shown in FIG. 16C was obtained. Collectedpeptide fractions were sequenced to identify the C-terminal peptide. Apeptide eluting at 36.8 min represents the C-terminal peptide. ThesequenceAsp-Ser-Arg-Val-Ser-Val-(X)-Lys-Pro-Phe-Phe-Met-Leu-Pro-Pro-Val-Ala-(Ala)(SEQ ID NO: 94) was assigned, where (X) denotes an unassigned residue,and (Ala) denotes tentative assignment due to low recovery. Theindicated amino acids corresponds to position 148-165 of the sequenceshown in FIG. 42.

After treatment of the C-terminal peptide with neuraminidase andO-glycanase to remove carbohydrate, fast atom bombardment-massspectroscopy (FAB-MS) analysis indicated a molecular weight of 1815.19for the protonated monoisotopic ion (NH⁺), consistent with the sequenceAsp-Ser-Arg-Val-Ser-Val-Thr-Lys-Pro-Phe-Phe-Met-Leu-Pro-Pro-Val-Ala-Ala(SEQ ID NO:95) (calculated molecular weight of MH⁺=1815.98). A lessabundant ion species of mass 1744.37, corresponding to theabove-mentioned peptide truncated by one Ala at the C-terminus(calculated MH⁺−1744.17), was also detected.

Further analyses were performed using electrospray mass spectroscopy(ES-MS). The deglycosylated C-terminal peptide fraction of the CHOcell-derived SCF and the C-terminal peptide fraction from E.coli-derived SCF¹⁻¹⁶⁵ (obtained as described in Example 2) wereanalyzed. A major signal with mass 1815 and a second signal with mass1743 were detected for the peptide of CHO cell-derived SCF. Only an 1814signal was detected for the peptide of E. coli-derived SCF.

These data indicate that soluble SCF is released from CHO cellstransfected with pDSRα2-Δ23 by proteolytic cleavage after amino acid 164or 165. This processing matches that found for BRL-3A cell derived ratSCF (Example 2).

EXAMPLE 6 Expression of Recombinant SCF in E. coli

A. Recombinant Rat SCF

This example relates to expression in E. coli of SCF polypeptides bymeans of a DNA sequence encoding [Met⁻¹] rat SCF¹⁻¹⁹³ (FIG. 14C).Although any suitable vector may be employed for protein expressionusing this DNA, the plasmid chosen was pCFM1156 (FIG. 19). This plasmidcan be readily constructed from pCFM 836 (see U.S. Pat. No. 4,710,473hereby incorporated by reference) by destroying the two endogenous NdeIrestriction sites by end-filling with T4 polymerase enzyme followed byblunt end ligation and substituting the small DNA sequence between theunique ClaI and KpnI restriction sites with the small oligonucleotideshown below.

5′ CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′ (SEQ IDNO:96)

3′ TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′ (SEQ ID NO:97)

Control of protein expression in the pCFM1156 plasmid is by means of asynthetic lambda P_(L) promoter which is itself under the control of atemperature sensitive lambda CI857 repressor gene [such as is providedin E. coli strains FM5 (ATCC deposit #53911) or K12ΔHtrp]. The pCFM1156vector is constructed so as to have a DNA sequence containing anoptimized ribosome binding site and initiation codon immediately 3′ ofthe synthetic PL promoter. A unique NdeI restriction site, whichcontains the ATG initiation codon, precedes a multi-restriction sitecloning cluster followed by a lambda t-oop transcription stop sequence.

Plasmid V19.8 SCF¹⁻¹⁹³ containing the rat SCF¹⁻¹⁹³ gene cloned from PCRamplified cDNA (FIG. 14C) as described in Example 3 was digested withBglII and SstII and a 603 bp DNA fragment isolated. In order to providea Met initiation codon and restore the codons for the first three aminoacid residues (Gln, Glu, and Ile) of the rat SCF polypeptide, asynthetic oligonucleotide linker

5′ TATGCAGGA (SEQ ID NO: 98) 3′

3′ ACGTCCTCTAG (SEQ ID NO:99) 5′

with NdeI and BglII sticky ends was made. The small oligonucleotide andrat SCF¹⁻¹⁹³ gene fragment were inserted by ligation into pCFM1156 atthe unique NdeI and SstII sites in the plasmid shown in FIG. 19. Theproduct of this reaction is an expression plasmid, pCFM1156 ratSCF¹⁻¹⁹³.

The pCFM1156 rat SCF¹⁻¹⁹³ plasmid was transformed into competent FM5 E.coli host cells. Selection for plasmid-containing cells was on the basisof the antibiotic (kanamycin) resistance marker gene carried on thepCFM1156 vector. Plasmid DNA was isolated from cultured cells and theDNA sequence of the synthetic oligonucleotide and its junction to therat SCF gene confirmed by DNA sequencing.

To construct the plasmid pCFM1156 rat SCF¹⁻¹⁶² encoding the [Met⁻¹] ratSCF¹⁻¹⁶² polypeptide, an EcoRI to SstII restriction fragment wasisolated from V19.8 rat SCF¹⁻¹⁶² and inserted by ligation into theplasmid pCFM rat SCF¹⁻¹⁹³ at the unique EcoRI and SstII restrictionsites thereby replacing the coding region for the carboxyl terminus ofthe rat SCF gene.

To construct the plasmids pCFM1156 rat SCF¹⁻¹⁶⁴ and pCFM1156 ratSCF¹⁻¹⁶⁵ encoding the [Met⁻¹] rat SCF¹⁻¹⁶⁴ and [Met⁻¹] rat SCF¹⁻¹⁶⁵polypetides, respectively, EcoRI to SstII restriction fragments wereisolated from PCR amplified DNA encoding the 3′ end of the SCF gene anddesigned to introduce site directed changes in the DNA in the regionencoding the carboxyl terminus of the SCF gene. The DNA amplificationswere performed using the oligonucleotide primers 227-29 and 237-19 inthe construction of pCFM1156 rat SCF¹⁻¹⁶⁴ and 227-29 and 237-20 in theconstruction of pCFM1156 rat SCF¹⁻¹⁶⁵.

B. Recombinant Human SCF

This example relates to the expression in E. coli of human SCFpolypeptide by means of a DNA sequence encoding [Met⁻¹] human SCF¹⁻¹⁶⁴and [Met⁻¹] human SCF¹⁻¹⁸³ (FIG. 15C); and [Met⁻¹] human SCF¹⁻¹⁶⁵ (FIG.15C). Plasmid V19.8 human SCF¹⁻¹⁶² containing the human SCF¹⁻¹⁶² genewas used as template for PCR amplification of the human SCF gene.Oligonucleotide primers 227-29 and 237-19 were used to generate the PCRDNA which was then digested with PstI and SstII restrictionendonucleases. In order to provide a Met initiation codon and restorethe codons for the first four amino acid residues (Glu, Gly, Ile, Cys)of the human SCF polypeptide, a synthetic oligonucleotide linker

5′ TATGGAAGGTATCTGCA (SEQ ID NO:100) 3′

3′ACCTTCCATAG (SEQ ID NO:101) 5′

with NdeI and PstI sticky ends was made. The small oligo linker and thePCR derived human SCF gene fragment were inserted by ligation into theexpression plasmid pCFM1156 (as described previously) at the unique NdeIand SstII sites in the plasmid shown in FIG. 19.

The pCFM1156 human SCF¹⁻¹⁶⁴ plasmid was transformed into competent FM5E. coli host cells. Selection for plasmid containing cells was on thebasis of the antibiotic (kanamycin) resistance marker gene carried onthe pCFM1156 vector. Plasmid DNA was isolated from cultured cells andthe DNA sequence of the human SCF gene confirmed by DNA sequencing.

To construct the plasmid pCFM1156 human SCF¹⁻¹⁸³ encoding the [Met⁻¹]human SCF¹⁻¹⁸³ (FIG. 15C) polypeptide, a EcoRI to HindIII restrictionfragment encoding the carboxyl terminus of the human SCF gene wasisolated from pGEM human SCF¹¹⁴⁻¹⁸³ (described below), a SstI to EcoRIrestriction fragment encoding the amino terminus of the human SCF genewas isolated from pCFM1156 human SCF¹⁻¹⁶⁴, and the larger HindIII toSstI restriction fragment from pCFM1156 was isolated. The three DNAfragments were ligated together to form the pCFM1156 human SCF¹⁻¹⁸³plasmid which was then tranformed into FM5 E. coli host cells. Aftercolony selection using kanamycin drug resistance, the plasmid DNA wasisolated and the correct DNA sequence confirmed by DNA sequencing. ThepGEM human SCF¹¹⁴⁻¹⁸³ plasmid is a derivative of pGEM3 that contains anEcoRI-SphI fragment that includes nucleotides 609 to 820 of the humanSCF cDNA sequence shown in FIG. 15C. The EcoRI-SphI insert in thisplasmid was isolated from a PCR that used oligonucleotide primers 235-31and 241-6 (FIG. 12B) and PCR 22.7 (FIG. 13B) as template. The sequenceof primer 241-6 was based on the human genomic sequence to the 3′ sideof the exon containing the codon for amino acid 176.

A plasmid encoding human [Met⁻¹] SCF¹⁻¹⁶⁵ was constructed as follows.Sixteen oligonucleotides were “stitched together” to create a 221 basepair fragment with EcoRl and BamHl sticky ends (FIG. 16D). Thisnucleotide sequence codes for the C-terminal 68 amino acids of humanSCF¹⁻¹⁸³ (amino acid numbering and designation as in FIG. 15C). Thecodons in this nucleotide sequence reflected those most commonly used byE. coli (i.e., optimized for expression in E. coli). In addition, aunique BstEII site is present in the fragment. The EcoRl to BamHlfragment of the human SCF¹⁻¹⁸³ DNA (FIG. 15C) was removed and replacedby the fragment containing the optimized codons. This construct wasdigested with BstEII and BamHl and the 39 base pair fragment shown inFIG. 16E was introduced. The resulting plasmid codes for human [Met⁻¹]SCF¹⁻¹⁶⁵ with the codons for the C-terminal 50 amino acis optimized forexpression in E. coli.

Another plasmid encoding human [Met⁻¹] SCF¹⁻¹⁶⁵, with the codons of FIG.15C, was also constructed, by PCR utilizing pCFM1156 human SCF¹⁻¹⁶⁴. A5′ oligonucleotide was made 5′ of the EcoRl site and a 3′oligonucleotide was made which included the final codons of the 1-164sequence plus an extra codon for the position 165 and nucleotidesthrough the SstII site. After the PCR reaction, the fragment was cutwith EcoRl and SstII, gel purified, and cloned into pCFM1156 humanSCF¹⁻¹⁶⁴ cut with EcoRl and SstII.

The generation of other expression plasmids including those encodinghuman [Met⁻¹] SCF¹⁻²⁴⁸ (sequence of FIG. 42) and encoding human [Met⁻¹]SCF¹⁻²²⁰ (sequence of FIG. 44) is described in Example 28.

C. Fermentation of E. coli producing Human SCF¹⁻¹⁶⁴ and E. coliproducing Human SCF¹⁻¹⁶⁵

Fermentations for the production of SCF¹⁻¹⁶⁴ were carried out in 16liter fermentors using an FM5 E. coli K12 host containing the plasmidpCFM 1156 human SCF¹⁻¹⁶⁴. Seed stocks of the producing culture weremaintained at −80° C. in 17% glycerol in Luria broth. For inoculumproduction, 100 μl of the thawed seed stock was transferred to 500 ml ofLuria broth in a 2 L erlenmeyer flask and grown overnight at 30° C. on arotary shaker (250 RPM).

For the production of E. coli cell paste used as starting material forthe purification of human SCF¹⁻¹⁶⁴ outlined in Example 10, the followingfermentation conditions were used.

The inoculum culture was aseptically transferred to a 16 L fermentorcontaining 8 L of batch medium (see Table 9). The culture was grown inbatch mode until the OD-600 of the culture was approximately 3-5. Atthis time, a sterile feed (Feed 1, Table 10) was introduced into thefermentor using a peristaltic pump to control the feed rate. The feedrate was increased exponentially with time to give a growth rate of 0.15hr⁻¹. The temperature was controlled at 30° C. during the growth phase.The dissolved oxygen concentration in the fermentor was automaticallycontrolled at 50% saturation using air flow rate, agitation rate, vesselback pressure and oxygen supplementation for control. The pH of thefermentor was automatically controlled at 7.0 using phosphoric acid andammonium hydroxide. At an OD-600 of approximately 30, the productionphase of the fermentation was induced by increasing the fermentortemperature to 42° C. At the same time the addition of Feed 1 wasstopped and the addition of Feed 2 (Table 11) was started at a rate of200 ml/hr. Approximately six hours after the temperature of thefermentor was increased, the fermentor contents were chilled to 15° C.The yield of SCF¹⁻¹⁶⁴ was approximately 30 mg/OD-L. The cell pellet wasthen harvested by centrifugation in a Beckman J6-B rotor at 3000×g forone hour. The harvested cell paste was stored frozen at −70° C.

An advantageous method for production of SCF¹⁻¹⁶⁴ is similar to themethod described above except for the following modifications.

1) The addition of Feed 1 is not initiated until the OD-600 of theculture reaches 5-6.

2) The rate of addition of Feed 1 is increased more slowly, resulting ina slower growth rate (approximately 0.08).

3) The culture is induced at OD-600 of 20.

4) Feed 2 is introduced into the fermentor at a rate of 300 mL/hr.

All other operations are similar to the method described above,including the media.

Using this process, yields of SCF¹⁻¹⁶⁴ approximately 35-40 mg/OD-L atOD=25 have been obtained.

TABLE 9 Composition of Batch Medium Yeast extract 10^(a) g/L Glucose 5K₂HPO₄ 3.5 KH₂PO₄ 4 M_(G)SO₄.7H₂O 1 NaCl 0.625 Dow P-2000 antifoam 5mL/8 L Vitamin solution^(b) 2 mL/L Trace metals solution^(c) 2 mL/L^(a)Unless otherwise noted, all ingredients are listed as g/L. ^(b)TraceMetals solution: FeCl₃.6H₂O, 27 g/L; ZnCl₂.4H₂O, 2 g/L; CaCl₂.6H₂O, 2g/L; Na₂MoO₄.2 H₂O, 2 g/L, CuSo₄.5 H₂O, 1.9 g/L; concentrated HCl, 100ml/L. ^(c)Vitamin solution: riboflavin, 0.42 g/l; pantothenic acid, 5.4g/L; niacin, 6 g/L; pyridoxine, 1.4 g/L; biotin, 0.06 g/L; folic acid,0.04 g/L.

TABLE 10 Composition of Feed Medium Yeast extract 50^(a) Glucose 450MgSO₄.7H₂O 8.6 Trace metals solution^(b) 10 mL/L Vitamin solution^(c) 10mL/L ^(a)Unless otherwise noted, all ingredients are listed as g/L.^(b)Trace Metals solution: FeCl₃.6H₂O, 27 g/L; ZnCl₂.4H₂O, 2 g/L;CaCl₂.6H₂O, 2 g/L; Na₂MoO₄.2 H₂O, 2 g/L, CuSo₄.5 H₂O, 1.9 g/L;concentrated HCl, 100 ml/L. ^(c)Vitamin solution: riboflavin, 0.42 g/l;pantothenic acid, 5.4 g/L; niacin, 6 g/L; pyridoxine, 1.4 g/L; biotin,0.06 g/L; folic acid, 0.04 g/L.

TABLE 11 Composition of Feed Medium 2 Tryptone 172^(a) Yeast extract  86Glucose 258 ^(a)All ingredients are listed as g/L.

For the production of E. coli cell paste used as starting material forthe purification of human SCF¹⁻¹⁶⁵ (Example 10), fermentation conditionsdiffered in the following ways from those described for the SCF¹⁻¹⁶⁴cases. Feed 1 was introduced when the OD-600 of the culture wasapproximately 5-6. Feed 1 contained 13 g/L K₂HPO₄ in addition to thecomponents listed in Table 10. The feed rate was increased exponentiallywith time to give a growth rate of 0.2 hr⁻¹. Production phase wasinduced by temperature increase at OD-600 of about 40, and the rate ofaddition of Feed 2 was 600 ml/hr. Feed 2 contained 258 g/L tryptone, 129g/L yeast extract, 50 g/L glucose, and 6.4 g/L K₂HPO₄. Chilling of thefermentor and harvesting of cells was done about eight hours after thetemperature increase.

EXAMPLE 7 Immunoassays for Detection of SCF

Radioimmunoassay (RIA) procedures applied for quantitative detection ofSCF in samples were conducted according to the following procedures.

An SCF preparation from BRL 3A cells purified as in Example 1 wasincubated together with antiserum for two hours at 37° C. After the twohour incubation, the sample tubes were then cooled on ice, ¹²⁵I-SCF wasadded, and the tubes were incubated at 4° C. for at least 20 h. Eachassay tube contained 500 μl of incubation mixture consisting of 50 μl ofdiluted antisera, ˜60,000 5 μl trasylol and 0-400 μl of SCF standard,with buffer (phosphate buffered saline, 0.1% bovine serum albumin, 0.05%Triton X-100, 0.025% azide) making up the remaining volume. Theantiserum was the second test bleed of a rabbit immunized with a 50%pure preparation of natural SCF from BRL 3A conditioned medium. Thefinal antiserum dilution in the assay was 1:2000.

The antibody-bound ¹²⁵I-SCF was precipitated by the addition of 150 μlStaph A (Calbiochem). After a 1 h incubation at room temperature, thesamples were centrifuged and the pellets were washed twice with 0.75 ml10 mM Tris-HCL pH 8.2, containing 0.15M NaCl, 2 mM EDTA, and 0.05%Triton X-100. The washed pellets were counted in a gamma counter todetermine the percent of ¹²⁵I-SCF bound. Counts bound by tubes lackingserum were subtracted from all final values to correct for nonspecificprecipitation. A typical RIA is shown in FIG. 20. The percent inhibitionof ¹²⁵I-SCF binding produced by the unlabeled standard is dose dependent(FIG. 20A), and, as indicated in FIG. 20B, when the immune precipitatedpellets are examined by SDS-PAGE and autoradiography, the ¹²⁵I-SCFprotein band is competed. In FIG. 20B, lane 1 is ¹²⁵I-SCF, and lanes 2,3, 4 and 5 are immune-precipicated ¹²⁵I-SCF competed with 0, 2, 100, and200 ng of SCF standard, respectively. As determined by both the decreasein antibody-precipitable cpm observed in the RIA tubes and decrease inthe immune-precipitated ¹²⁵I-SCF protein band (migrating atapproximately M_(r) 31,000) the polyclonal antisera recognizes the SCFstandard which was purified as in Example 1.

Western procedures were also applied to detect recombinant SCF expressedin E. coli, COS-1, and CHO cells. Partially purified E. coli expressedrat SCF¹⁻¹⁹³ (Example 10), COS-1 cell expressed rat SCF¹⁻¹⁶² andSCF¹⁻¹⁹³ as well as human SCF¹⁻¹⁶² (Examples 4 and 9), and CHO cellexpressed rat SCF¹⁻¹⁶² (Example 5), were subjected to SDS-PAGE.Following electrophoresis, the protein bands were transferred to 0.2 μmnitrocellulose using a Bio-Rad Transblot apparatus at 60V for 5 h. Thenitrocellulose filters were blocked for 4 h in PBS, pH 7.6, containing10% goat serum followed by a 14 h room temperature incubation with a1:200 dilution of either rabbit preimmune or immune serum (immunizationdescribed above). The antibody-antiserum complexes were visualized usinghorseradish peroxidase-conjugated goat anti-rabbit IgG reagents (Vectorlaboratories) and 4-chloro-1-napthol color development reagent.

Examples of two Western analyses are presented in FIGS. 21 and 22. InFIG. 21, lanes 3 and 5 are 200 μl of COS-1 cell produced human SCF¹⁻¹⁶²;lanes 1 and 7 are 200 μl of COS-1 cell produced human EPO (COS-1 cellstransfected with V19.8 EPO); and lane 8 is prestained molecular weightmarkers. Lanes 1-4 were incubated with pre-immune serum and lanes 5-8were incubated with immune serum. The immune serum specificallyrecognizes a diffuse band with an apparent M_(r) of 30,000 daltons fromCOS-1 cells producing human SCF¹⁻¹⁶² but not from COS-1 cells producinghuman EPO.

In the Western shown in FIG. 22, lanes 1 and 7 are 1 μg of a partiallypurified preparation of rat SCF¹⁻¹⁹³ produced in E. coli; lanes 2 and 8are wheat germ agglutinin-agarose purified COS-1 cell produced ratSCF¹⁻¹⁹³; lanes 4 and 9 are wheat germ agglutinin-agarose purified COS-1cell produced rat SCF¹⁻¹⁶²; lanes 5 and 10 are wheat germagglutinin-agarose purified CHO cell produced rat SCF¹⁻¹⁶²; and lane 6is prestained molecular weight markers. Lanes 1-5 and lanes 6-10 wereincubated with rabbit preimmune and immune serum, respectively. The E.coli produced rat SCF¹⁻¹⁹³ (lanes 1 and 7) migrates with an apparentM_(r) of ˜24,000 daltons while the COS-1 cell produced rat SCF¹⁻¹⁹³(lanes 2 and 8) migrates with an apparent M_(r) of 24-36,000 daltons.

This difference in molecular weights is expected since mammalian cells,but not bacteria, are capable of glycosylation. Transfection of thesequence encoding rat SCF¹⁻¹⁶² into COS-1 (lanes 4 and 9), or CHO cells(lanes 5 and 10), results in expression of SCF with a lower averagemolecular weight than that produced by transfection with SCF¹⁻¹⁹³ (lanes2 and 8).

The expression products of rat SCF¹⁻¹⁶² from COS-1 and CHO cells are aseries of bands ranging in apparent M_(r) between 24-36,000 daltons. Theheterogeneity of the expressed SCF is likely due to carbohydratevariants, where the SCF polypeptide is glycosylated to differentextents.

In summary, Western analyses indicate that immune serum from rabbitsimmunized with natural mammalian SCF recognize recombinant SCF producedin E. coli, COS-1 and CHO cells but fail to recognize any bands in acontrol sample consisting of COS-1 cell produced EPO. In further supportof the specificity of the SCF antiserum, preimmune serum from the samerabbit failed to react with any of the rat or human SCF expressionproducts.

Radioimmunoasssaay (RIA) procedures were also developed to quantify SCFin human serum samples. Purified CHO-derived human SCF (expression ofthe 1-248 transcript) was used as the standard in this assay over therange of 0.01-10.0 ng/tube. Pooled normal human serum samples, obtainedfrom Irvine Scientific (Lots 500080713 and 500081015), were each assayedat 25, 50, 100 and 200 μl per tube. Each tube was adjusted to contain 5μl of trasylol, and 900 μl total volume by the addition of theappropriate amount of assay diluent (phosphate-buffered salinecontaining 0.1% bovine serum albumin and 0.025% sodium azide). Rabbitanti-human SCF antiserum (100 μl of a 1:50,000 dilution) was added, thetubes were mixed and incubated at 4° C. for approximately 24 hours. Theantiserum was the bleed-out of a rabbit hyperimmunized with a purifiedpreparation of CHO-derived human SCF¹⁻¹⁶².

Following the 24 hours incubation, approximately 60,000 cpm of¹²⁵I-CHO-derived human SCF (expression of the 1-248 transcript, 57.9mCi/mg) was added to all tubes; the tubes were vortexed and incubated at4° C. for an additional 19 hours. The antibody-bound ¹²⁵I-human SCF wasprecipitated by the addition of 100 μl of a 1:50 dilution of normalrabbit serum (Research Products International) and 100 μl of a 1:20dilution of goat anti-rabbit IgG (Research Products International) toall tubes. After a two hour incubation at room temperature, the tubeswere centrifuged and the pellets were washed once with 0.75 ml of 10 mMTris-HCl, pH 8.2, containing 0.15 M NaCl, 2 mM EDTA, and 0.05% TritonX-100. The washed pellets were counted in a gamma counter to determinethe percent of ¹²⁵I-human SCF bound. Counts bound by tubes lackingantiserum were subtracted from all final values to correct fornonspecific precipitation. A typical RIA is shown in FIG. 22A. Thepercent inhibition of ¹²⁵I-human SCF binding by the unlabeled standardand normal human serum was dose-dependent. Increasing aliquots of thenormal human serum, over the range of 25-200 μl produced a dose responseline which was parallel to that of the standard. Both of the normalhuman serum samples were assayed twice in this assay. Values plotted inFIG. 22A are the average percent inhibitions obtained for the respectivealiquots for each serum sample. Values of 2.16 ng/ml and 2.93 ng/ml wereobtained for SCF levels in Lot 500080713 and Lot 500081015 normal humanserum, respectively.

EXAMPLE 8 In Vivo Activity of Recombinant SCF

A. Rat SCF in Bone Marrow Transplanation

COS-1 cells were transfected with V19.8 SCF¹⁻¹⁶² in a large scaleexperiment (T175 cm² flasks instead of 60 mm dishes) as described inExample 4. Approximately 270 ml of supernatant was harvested. Thissupernatant was chromatographed on wheat germ agglutinin-agarose andS-Sepharose essentially as described in Example 1. The recombinant SCFwas evaluated in a bone marrow transplantation model based on murineW/W^(v) genetics. The W/W^(v) mouse has a stem cell defect which amongother features results in a macrocytic anemia (large red cells) andallows for the transplantation of bone marrow from normal animalswithout the need for irradiation of the recipient animals [Russel, etal., Science, 144, 844-846 (1964)]. The normal donor stem cells outgrowthe defective recipient cells after transplantation.

In the following example, each group contained six age matched mice.Bone marrow was harvested from normal donor mice and transplanted intoW/W^(v) mice. The blood profile of the recipient animals is followed atdifferent times post transplantation and engraftment of the donor marrowis determined by the shift of the peripheral blood cells from recipientto donor phenotype. The conversion from recipient to donor phenotype isdetected by monitoring the forward scatter profile (FASCAN, BectonDickenson) of the red blood cells. The profile for each transplantedanimal was compared to that for both donor and recipient untransplantedcontrol animals at each time point. The comparison was made utilizing acomputer program based on Kolmogorov-Smirnov statistics for the analysisof histograms from flow systems [Young, J. Histochem. and Cytochem., 25,935-941 (1977)]. An independent qualitative indicator of engraftment isthe hemoglobin type detected by hemoglobin electrophoresis of therecipient blood [Wong, et al., Mol. and Cell. Biol., 9, 798-808 (1989)]and agrees well with the goodness of fit determination fromKolmogorov-Smirnov statistics.

Approximately 3×10⁵ cells were transplanted without SCF treatment(control group in FIG. 23) from C56BL/6J donors into W/W^(v) recipients.A second group received 3×10⁵ donor cells which had been treated withSCF (600 U/ml) at 37° C. for 20 min and injected together (pre-treatedgroup in FIG. 23). (One unit of SCF is defined as the amount whichresults in half-maximal stimulation in the MC/9 bioassay). In a thirdgroup, the recipient mice were injected sub-cutaneously (sub-Q) withapproximately 400 U SCF/day for 3 days after transplantation of 3×10⁵donor cells (Sub-Q inject group in FIG. 23). As indicated in FIG. 23, inboth SCF-treated groups the donor marrow is engrafted faster than in theuntreated control group. By 29 days post-transplantation, the SCFpre-treated group had converted to donor phenotype. This Exampleillustrates the usefulness of SCF therapy in bone marrowtransplantation.

B. In Vivo Activity of Rat SCF in Steel Mice

Mutations at the S1 locus cause deficiencies in hematopoietic cells,pigment cells, and germ cells. The hematopoietic defect is manifest asreduced numbers of red blood cells [Russell, In:Al Gordon, Regulation ofHematopoiesis, Vol. I, 649-675 Appleton-Century-Crafts, New York(1970)], neutrophils [Ruscetti, Proc. Soc. Exp. Biol. Med., 152, 398(1976)], monocytes [Shibata, J. Immunol. 135, 3905 (1985)],megakaryocytes [Ebbe, Exp. Hematol., 6, 201 (1978)], natural killercells [(Clark, Immunogenetics, 12, 601 (1981)], and mast cells [Hayashi,Dev. Biol., 109, 234 (1985)]. Steel mice are poor recipients of a bonemarrow transplant due to a reduced ability to support stem cells[Bannerman, Prog. Hematol., 8, 131 (1973)]. The gene encoding SCF isdeleted in Steel (S1/S1) mice.

Steel mice provide a sensitive in vivo model for SCF activity. Differentrecombinant SCF proteins were tested in Steel-Dickie (S1/S1^(d)) micefor varying lengths of time. Six to ten week old Steel mice(WCB6F1-S1/S1^(d)) were purchased from Jackson Labs, Bar Harbor, Me.Peripheral blood was monitored by a SYSMEX F-800 microcell counter(Baxter, Irvine, Calif.) for red cells, hemoglobin, and platelets. Forenumeration of peripheral white blood cell (WBC) numbers, a CoulterChannelyzer 256 (Coulter Electronics, Marietta, Ga.) was used.

In the experiment in FIG. 24, Steel-Dickie mice were treated with E.coli derived SCF¹⁻¹⁶⁴, purified as in Example 10, at a dose of 100μg/kg/day for 30 days, then at a dose of 30 μg/kg/day for an additional20 days. The protein was formulated in injectable saline (Abbott Labs,North Chicago, Ill.) +0.1% fetal bovine serum. The injections wereperformed daily, subcutaneously. The peripheral blood was monitored viatail bleeds of ˜50 μl at the indicated times in FIG. 24. The blood wascollected into 3% EDTA coated syringes and dispensed into powdered EDTAmicrofuge tubes (Brinkmann, Westbury, N.Y.). There is a significantcorrection of the macrocytic anemia in the treated animals relative tothe control animals. Upon cessation of treatment, the treated animalsreturn to the initial state of macrocytic anemia.

In the experiment shown in FIG. 25 and 26, Steel-Dickie mice weretreated with different recombinant forms of SCF as described above, butat a dose of 100 μg/kg/day for 20 days. Two forms of E. coli derived ratSCF, SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁹³, were produced as described in Example 10. Inaddition, E. coli SCF¹⁻¹⁶⁴, modified by the addition of polyethyleneglycol (SCF¹⁻¹⁶⁴ PEG25) as in Example 12, was also tested. CHO derivedSCF¹⁻¹⁶² produced as in Example 5 and purified as in Example 11, wasalso tested. The animals were bled by cardiac puncture with 3% EDTAcoated syringes and dispensed into EDTA powdered tubes. The peripheralblood profiles after 20 days of treatment are shown in FIG. 25 for whiteblood cells (WBC) and FIG. 26 for platelets. The WBC differentials forthe SCF¹⁻¹⁶⁴ PEG25 group are shown in FIG. 27. There are absoluteincreases in neutrophils, monocytes, lymphocytes, and platelets. Themost dramatic effect is seen with SCF¹⁻¹⁶⁴ PEG 25.

An independent measurement of lymphocyte subsets was also performed andthe data is shown in FIG. 28. The murine equivalent of human CD4, ormarker of T helper cells, is L3T4 [Dialynas, J. Immunol., 131, 2445(1983)]. LyT-2 is a murine antigen on cytotoxic T cells [Ledbetter, J.Exp. Med., 153, 1503 (1981)]. Monoclonal antibodies against theseantigens were used to evaluate T cell subsets in the treated animals.

Whole blood was stained for T lymphocyte subsets as follows. Two hundredmicroliters of whole blood was drawn from individual animals into EDTAtreated tubes. Each sample of blood was lysed with sterile deionizedwater for 60 seconds and then made isotonic with 10× Dulbecco'sPhosphate Buffered Saline (PBS) (Gibco, Grand Island, N.Y.). This lysedblood was washed 2 times with 1× PBS (Gibco, Grand Island, N.Y.)supplemented with 0.1% Petal Bovine Serum (Flow Laboratory, McLean, Va.)and 0.1% sodium azide. Each sample of blood was deposited into roundbottom 96 well cluster dishes and centrifuged. The cell pellet(containing 2-10×10⁵ cells) was resuspended with 20 microliters of Ratanti-Mouse L3T4 conjugated with phycoerythrin (PE) (Becton Dickinson,Mountain View, Calif.) and 20 microliters of Rat anti-Mouse Lyt-2conjugated with Fluorescein Isothiocyanate incubated on ice (4° C.) for30 minutes (Becton Dickinson). Following incubation the cells werewashed 2 times in 1× PBS supplemented as indicated aboved. Each sampleof blood was then analyzed on a FACScan cell analysis system (BectonDickinson, Mountain View, Calif.). This system was standardized usingstandard autocompensation procedures and Calibrite Beads (BectonDickinson, Mountain View, Calif.). These data indicated an absoluteincrease in both helper T cell populations as well as cytotoxic T cellnumbers.

C. In Vivo Activity of SCF in Primates

Human SCF¹⁻¹⁶⁴ expressed in E. coli (Example 6B) and purified tohomogeneity as in Example 10, was tested for in vivo biological activityin normal primates. Adult male baboons (Papio sp.) were studied in threegroups: untreated, n=3; SCF 100 ug/kg/day, n=6; and SCF 30 ug/kg/day,n=6. The treated animals received single daily subcutaneous injectionsof SCF. Blood specimens were obtained from the animals under ketaminerestraint. Specimens for complete blood count, reticulocyte count, andplatelet count were obtained on days 1, 6, 11, 15, 20 and 25 oftreatment.

All animals survived the protocol and had no adverse reactions to SCFtherapy. The white blood cell count increased in the 100 ug/kg treatedanimals as depicted in FIG. 29. The differential count, obtainedmanually from Wright Giemsa stained peripheral blood smears, is alsoindicated in FIG. 29. There was an absolute increase in neutrophils,lymphocytes, and monocytes. As indicated in FIG. 30 there was also anincrease at the 100 ug/kg dose in the hemtocrits as well as platelets.

Human SCF (hSCF¹⁻¹⁶⁴ modified by the addition of polyethylene glycol asin Example 12) was also tested in normal baboons, at a dose of 200μg/kg-day, administered by continuous intravenous infusion and comparedto the unmodified protein. The animals started SCF at day 0 and weretreated for 28 days. The results for the peripheral WBC are given in thefollowing table. The PEG modified SCF elicited an earlier rise inperipheral WBC than the unmodified SCF. The same results are obtainedwith human SCF¹⁻¹⁶⁵ modified by the addition of polyethylene glycol.

Treatment with 200 μg/kg-day hSCF¹⁻¹⁶⁴:

Animal #M88320 Animal #M88129 DAY WBC DAY WBC 0 5800 0 6800 +7 10700 +77400 +14 12600 +14 20900 +16 22000 +21 18400 +22 31100 +23 24900 +24328100 +29 13000 +29 9600 +30 23000 +36 6600 +37 12100 +43 5600 +4410700 +51 7800

Treatment with 200 μg/kg-day PEG-hSCF¹⁻¹⁶⁴:

Animal # M88350 Animal # M89116 DAY WBC DAY WBC  −7 12400  −5  7900  −211600    0  7400  +4 24700  +6 16400  +7 20400  +9 17100 +11 24700 +1318700 +14 32600 +16 19400 +18 33600 +20 27800 +21 26400 +23 20700 +2516600 +27 20200 +28 26900 +29 18600 +32  9200 +33  7600

Human SCF¹⁻¹⁶⁵ expressed in E. coli (Example 6) and purified tohomogeneity as in Example 10B, demonstrates the same in vivo biologicalactivity in primotes as E. coli derived recombinant human SCF¹⁻¹⁶⁴.

EXAMPLE 9 In vitro Activity of Recombinant Human SCF

A. Human bone marrow assay, murine HPP-CFC assay, and murine MC/9 assay.

The cDNA of human SCF corresponding to amino acids 1-162 obtained by PCRreactions outlined in Example 3D, was expressed in COS-1 cells asdescribed for the rat SCF in Example 4. COS-1 supernatants were assayedon human bone marrow as well as in the murine HPP-CFC and MC/9 assays.The human protein was not active at the concentrations tested in eithermurine assay; however, it was active on human bone marrow. The cultureconditions of the assay were as follows: human bone marrow from healthyvolunteers was centrifuged over Ficoll-Hypaque gradients (Pharmacia) andcultured in 2.1% methyl cellulose, 30% fetal calf serum, 6×10⁻⁵ M2-mercaptoethanol, 2 mM glutamine, ISCOVE'S medium (GIBCO), 20 U/ml EPO,and 1×10⁵ cells/ml for 14 days in a humidified atmosphere containing 7%O₂, 10% CO₂, and 83% N₂. The colony numbers generated with recombinanthuman and rat SCF COS-1 supernatants are indicated in Table 12. Onlythose colonies of 0.2 mm in size or larger are indicated.

TABLE 12 Growth of Human Bone Marrow Colonies in Response to SCF Volumeof CM Colony #/100,000 Plasmid Transfected Assayed (μl) cells ± SD V19.8(no insert) 100 0  50 0 V19.8 human SCF¹⁻¹⁶² 100 33 ± 7  50 22 ± 3 V19.8rat SCF¹⁻¹⁶² 100 13 ± 1  50 10 

The colonies which grew over the 14 day period are shown in FIG. 31A(magnification 12×). The arrow indicates a typical colony. The coloniesresembled the murine HPP-CFC colonies in their large size (average 0.5mm). Due to the presence of EPO, some of the colonies werehemoglobinized. When the colonies were isolated and centrifuged ontoglass slides using a Cytospin (Shandon) followed by staining withWright-Giemsa, the predominant cell type was an undifferentiated cellwith a large nucleus:cytoplasm ratio as shown in FIG. 31B (magnification400×). The arrows in FIG. 31B point to the following structures: arrow1, cytoplasm; arrow 2, nucleus; arrow 3, vacuoles. Immature cells as aclass are large and the cells become progressively smaller as theymature [Diggs et al., The Morphology of Human Blood Cells, Abbott Labs,3 (1978)]. The nuclei of early cells of the hemotopoietic maturationsequence are relatively large in relation to the cytoplasm. In addition,the cytoplasm of immature cells stains darker with Wright-Giemsa thandoes the nucleus. As cells mature, the nucleus stains darker than thecytoplasm. The morphology of the human bone marrow cells resulting fromculture with recombinant human SCF is consistent with the conclusionthat the target and immediate product of SCF action is a relativelyimmature hematopoietic progenitor.

Recombinant human SCF was tested in agar colony assays on human bonemarrow in combination with other growth factors as described above. Theresults are shown in Table 13. SCF synergizes with G-CSF, GM-CSF, IL-3,and EPO to increase the proliferation of bone marrow targets for theindividual CSFs.

TABLE 13 Recombinant human SCF Synergy with Other Human ColonyStimulating Factors Colony #/10⁵ cells (14 Days) mock 0 hG-CSF 32 ± 3hG-CSF + hSCF 74 ± 1 hGM-CSF 14 ± 2 hGM-CSF + hSCF 108 ± 5  hIL-3 23 ± 1hIL-3 + hSCF 108 ± 3  hEPO 10 ± 5 hEPO + IL-3 17 ± 1 hEPO + hSCF  86 ±10 hSCF 0

Another activity of recombinant human SCF is the ability to causeproliferation in soft agar of the human acute myelogenous leukemia (AML)cell line, KG-1 (ATCC CCL 246). COS-1 supernatants from transfectedcells were tested in a KG-1 agar cloning assay [Koeffler et al.,Science, 200, 1153-1154 (1978)] essentially as described except cellswere plated at 3000/ml. The data from triplicate cultures are given inTable 14.

TABLE 14 KG-1 Soft Agar Cloning Assay Volume Colony #/3000 PlasmidTransfected Assayed (μl) Cells ± SD V19.8 (no insert) 25 2 ± 1 V19.8human SCF¹⁻¹⁶² 25 14 ± 0  12 8 ± 0 6 9 ± 5 3 6 ± 4 1.5 6 ± 6 V19.8 ratSCF¹⁻¹⁶² 25 6 ± 1 human GM-CSF 50 (5 ng/ml) 14 ± 5 

B. UT-7 ³H-Thymidine Uptake Assay

UT-7 cells are a human megakaryocyte, huGM-CSF responsive cell lineobtained from John Adamson, New York Blood Center, New York, N.Y. UT-7cells were cultured in Iscove's Modified Dulbecco's Medium, 10% FBS,1×glutamine, 5 \g/ml huGM-CSF. Cells are passaged twice a week at 1×10⁵cells/ml.

Cells were washed twice in phosphate buffered saline (PBS) andresuspended in RPMI medium with 4% FBS and glutamine penicillinstreptomycin (GPS) (Irvine Scientific Cat No. 9316 used at 1% volume pervolume) at 4×10⁴ cells/ml before use. Human SCF along with specificsamples were added to 4000 cells/well in 96 well plates and werecultured for 72 hrs. 0.5 uCi/well of ³H-Thymidine was then added to eachplate, plates were harvested and counted 4 hours later. A typical assayis shown in FIG. 31C.

Activity of human [Met⁻¹]SCF¹⁻¹⁶⁴ and human [Met⁻¹]SCF¹⁻¹⁶⁵, preparedfrom E. coli as described in Example 10, are also equally active instimulating the proliferation of the UT-7 cell line, as shown in FIG.31C.

C. SCF Radio-Receptor Assay Protocol

OCIM1 cells, [Papayannopoulou et al., Blood 72:1029-1038 (1988)] are ahuman erythroleukemic cell line expressing many human SCF receptors percell. These cells are grown in Iscove's Modified Dulbecco's Medium, 10%FBS, and 1× glutamine and passaged 3 times a week to 1×10⁵ cells/ml.

Preparation of the OCIM1 plasma membrane is as follows with all stepsperformed on ice.

First, 40 T175 flasks of cells were grown-up in OCIM1 culture medium,for a total of 1.9×10⁹ cells/ml. The conditioned medium and 1 mM PhenylMethyl Sulfonyl Fluoride (PMSF) protease inhibitor, was spun down in8×250 ml tubes at 1000 rpm for 10 minutes at 4° C. Cells were washedwith PBS and repelleted in 4×50 ml centrifuge tubes at 1000 rpm for 10minutes at 4° C. Cells were resuspended in 20 ml ice cold PBS withglucose sodium pyruvate (Gibco Cat #310-4287). The 20 ml cell solutionwas put into a pre-pressurized, pre-chilled (4° C.) “cell bomb” designedto lyse the cells. Cells were pressurized at 400-650 PSI for 10 minutesto establish equilibrium. When the pressure is released cell lysisoccurs.

At this point the cells were checked for the percentage of cell lysis.90% lysis was common. The cell suspension was resuspended in 80 mlssucrose buffer (0.25M sucrose, 10 mM Tris, 1 mM EDTA in double distilled(dd) H₂O, filtered through a 0.45 u filter, pH 7.0) and divided betweentwo 40 ml screwcap tubes. Tubes were spun at 5900 RPM for 10 minutes ina Beckman J2-21 centrifuge, JA-20 rotor at 4° C. The supernatants weresaved and spun one more time as above to further remove any unwantedmaterial. Supernatants were saved and distributed equally into 2 nalgene40 ml centrifuge tubes. These supernatants were centrifuged at 16,000RPM 4° C. for 30 min. in J2-21 centrifuge, JA-20 rotor. Thesesupernatants were discarded being careful to save pellets. Each pelletwas resuspended in sucrose buffer so there were 20 mls per tube in 4×36ml plastic ultracentrifuge tubes. Using a 20 ml syringe and a largetrochar, the solution was carefully underlayered in each tube with icecold 36% sucrose solution (36.1 g sucrose/100 mls ddH₂O), bringing thelevel of the liquid to within 2 mm of the top of the tube. Withoutdisturbing the interface, each tube was carefully placed into each of 6titanium ultracentrifuge tubes. Tubes were centrifuged at 27,000 RPM, 4°C. for 75 minutes in an ultracentrifuge. These tubes were carefullyremoved from the rotor and from titanium buckets, placed in a rack withthe 36% sucrose interface visible. The membraneous material at theinterface was collected with a pasteur pipet and transfered into 2 cleannalgene 40 ml centrifuge tubes. Volume was brought up to 40 mls with icecold sucrose buffer. Tubes were balanced and centrifuged as before at5900 RPM in J2-21 centrifuge. The supernatant was discarded and eachpellet was resuspended in 4 mls ice cold Tris buffer (10 mM Tris, 1 mMEDTA, pH 7.0 in ddH₂) with a 1 ml micropipet repeatedly, to ensurehomogeneity of the solutions. Storage was in 50 ul aliquots at −70° C.in freezing vials.

The SCF radioreceptor assay was conducted as follows with all stepsbeing performed on ice. Human SCF samples were diluted in RRA buffer (50mM Tris, 0.25% BSA pH 7.5) and added to 1.5 ml eppendorf tubes up to 150ul total volume. 50,000 counts in 50 ul buffer of ¹²⁵I-huSCF (providedby ICN radiochemicals) were added to each tube. A dilution of isolatedOCIM1 plasma membrane in 50 ul buffer known to give 20% specific bindingwas then added to each tube. Tubes were vortexed and allowed to incubatefor 24 hrs at 4° C. 400ul of buffer was then added to each tube and thetubes were centrifuged for 8 minutes at 18,000 RPM in J2-21 centrifuge,JA-18.1 fixed angle (45%) rotor, 4° C. All tubes were oriented with lidopening tabs straight up. Supernatants were carefully aspirated by asliding a 21 gauge needle down the side opposite the pellet (hinge sideof tube) to bottom of each tube. Tubes were counted in gamma counter for1 min. each.

In the radioreceptor assay, human [Met⁻¹]SCF¹⁻¹⁶⁴ and human[Met⁻¹]SCF¹⁻¹⁶⁵, prepared from E. coli as described in Example 10,compete equally well with the binding of human [¹²⁵I][[Met⁻¹]SCF¹⁻¹⁶⁴,indicating that they bind equally well to the SCF receptor.

EXAMPLE 10 Purification of Recombinant SCF Products Expressed in E. coli

A. SCF¹⁻¹⁶⁴

Fermentation of E. coli human SCF¹⁻¹⁶⁴ was performed according toExample 6C. The harvested cells (912 g wet weight) were suspended inwater to a volume of 4.6 L and broken by three passes through alaboratory homogenizer (Gaulin Model 15MR-8TBA) at 8000 psi. A brokencell pellet fraction was obtained by centrifugation (17700×g, 30 min, 4°C.), washed once with water (resuspension and recentrifugation), andfinally suspended in water to a volume of 400 ml.

The pellet fraction containing insoluble SCF (estimate of 10-12 g SCF)was added to 3950 ml of an appropriate mixture such that the finalconcentrations of components in the mixture were 8 M urea (ultrapuregrade), 0.1 mM EDTA, 50 mM sodium acetate, pH 6-7; SCF concentration wasestimated as 1.5 mg/ml. Incubation was carried out at room temperaturefor 4 h to solubilize the SCF. Remaining insoluble material was removedby centrifugation (17700×g, 30 min, room temperature). Forrefolding/reoxidation of the solubilized SCF, the supernatant fractionwas added slowly, with stirring, to 39.15 L of an appropriate mixturesuch that the final concentrations of components in the mixture were 2.5M urea (ultrapure grade), 0.01 mM EDTA, 5 mM sodium acetate, 50 mMTris-HCl pH 8.5, 1 mM glutathione, 0.02% (wt/vol) sodium azide. SCFconcentration was estimated as 150 μg/ml. After 60 h at room temperature[shorter times (e.g. ˜20 h) are suitable also], with stirring, themixture was concentrated two-fold using a Millipore Pelliconultrafiltration apparatus with three 10,000 molecular weight cutoffpolysulfone membrane cassettes (15 ft² total area) and then diafilteredagainst 7 volumes of 20 mM Tris-HCl, pH 8. The temperature during theconcentration/ultrafiltration was 4° C., pumping rate was 5 L/min, andfiltration rate was 600 ml/min. The final volume of recovered retentatewas 26.5 L. By the use of SDS-PAGE carried out both with and withoutreduction of samples, it is evident that most (>80%) of the pelletfraction SCF is solubilized by the incubation with 8 M urea, and thatafter the folding/oxidation multiple species (forms) of SCF are present,as visualized by the SDS-PAGE of unreduced samples. The major form,which represents correctly oxidized SCF (see below), migrates withapparent M_(r) of about 17,000 (unreduced) relative to the molecularweight markers (reduced) described for FIG. 9. Other forms includematerial migrating with apparent M_(r) of about 18-20,000 (unreduced),thought to represent SCF with incorrect intrachain disulfide bonds; andbands migrating with apparent M_(r)s in the range of 37,000 (unreduced),or greater, thought to represent various SCF forms having interchaindisulfide bonds resulting in SCF polypeptide chains that are covalentlylinked to form dimers or larger oligomers, respectively. The followingfractionation steps result in removal of remaining E. coli contaminantsand of the unwanted SCF forms, such that SCF purified to apparenthomogeneity, in biologically active conformation, is obtained.

The pH of the ultrafiltration retentate was adjusted to 4.5 by additionof 375 ml of 10% (vol/vol) acetic acid, leading to the presence ofvisible precipitated material. After 60 min, at which point much of theprecipitated material had settled to the bottom of the vessel, the upper24 L were decanted and filtered through a Cuno™ 30SP depth filter at 500ml/min to complete the clarification. The filtrate was then diluted1.5-fold with water and applied at 4° C. to an S-Sepharose Fast Flow(Pharmacia) column (9×18.5 cm) equilibrated in 25 mM sodium acetate, pH4.5. The column was run at a flow rate of 5 L/h, at 4° C. After sampleapplication, the column was washed with five column volumes (˜6 L) ofcolumn buffer and SCF material, which was bound to the column, waseluted with a gradient of 0 to 0.35 M NaCl in column buffer. Totalgradient volume was 20 L and fractions of 200 ml were collected. Theelution profile is depicted in FIG. 33. Aliquots (10 μl) from fractionscollected from the S-Sepharose column were analyzed by SDS-PAGE carriedout both with (FIG. 32 A) and without (FIG. 32 B) reduction of thesamples. From such analyses it is apparent that virtually all of theabsorbance at 280 nm (FIGS. 32 and 33) is due to SCF material.

The correctly oxidized form predominates in the major absorbance peak(fractions 22-38, FIG. 33). Minor species (forms) which can bevisualized in fractions include the incorrectly oxidized material withapparent M_(r) of 18-20,000 on SDS-PAGE (unreduced), present in theleading shoulder of the main absorbance peak (fractions 10-21, FIG. 32B); and disulfide-linked dimer material present throughout theabsorbance region (fractions 10-38, FIG. 32 B).

Fractions 22-38 from the S-Sepharose column were pooled, and the poolwas adjusted to pH 2.2 by addition of about 11 ml 6 N HCl and applied toa Vydac C₄ column (height 8.4 cm, diameter 9 cm) equilibrated with 50%(vol/vol) ethanol, 12.5 mM HCl (solution A) and operated at 4° C. Thecolumn resin was prepared by suspending the dry resin in 80% (vol/vol)ethanol, 12.5 mM HCl (solution B) and then equilibrating it withsolution A. Prior to sample application, a blank gradient from solutionA to solution B (6 L total volume) was applied and the column was thenre-equilibrated with solution A. After sample application, the columnwas washed with 2.5 L of solution A and SCF material, bound to thecolumn, was eluted with a gradient from solution A to solution B (18 Ltotal volume) at a flow rate of 2670 ml/h. 286 fractions of 50 ml eachwere collected, and aliquots were analyzed by absorbance at 280 nm (FIG.35), and by SDS-PAGE (25 μl per fraction) as described above (FIG. 34 A,reducing conditions; FIG. 34 B, nonreducing conditions). Fractions62-161, containing correctly oxidized SCF in a highly purified state,were pooled [the relatively small amounts of incorrectly oxidizedmonomer with M_(r) of about 18-20,000 (unreduced) eluted later in thegradient (about fractions 166-211) and disulfide-linked dimer materialalso eluted later (about fractions 199-235) (FIG. 35)].

To remove ethanol from the pool of fractions 62-161, and to concentratethe SCF, the following procedure utilizing Q-Sepharose Fast Flow(Pharamcia) ion exchange resin was employed. The pool (5 L) was dilutedwith water to a volume of 15.625 L, bringing the ethanol concentrationto about 20% (vol/vol). Then 1 M Tris base (135 ml) was added to bringthe pH to 8, followed by 1 M Tris-HCl, pH 8, (23.6 ml) to bring thetotal Tris concentration to 10 mM. Next 10 mM Tris-HCl, pH 8 (˜15.5 L)was added to bring the total volume to 31.25 L and the ethanolconcentration to about 10% (vol/vol). The material was then applied at4° C. to a column of Q-Sepharose Fast Flow (height 6.5 cm, diameter 7cm) equilibrated with 10 mM Tris-HCl, pH 8, and this was followed bywashing of the column with 2.5 L of column buffer. Flow rate duringsample application and wash was about 5.5 L/h. To elute the bound SCF,200 mM NaCl, 10 mM Tris-HCl, pH 8 was pumped in reverse directionthrough the column at about 200 ml/h. Fractions of about 12 ml werecollected and analyzed by absorbance at 280 nm, and SDS-PAGE as above.Fractions 16-28 were pooled (157 ml).

The pool containing SCF was then applied in two separate chromatographicruns (78.5 ml applied for each) to a Sephacryl S-200 HR (Pharmacia) gelfiltration column (5×138 cm) equilibrated with phosphate-buffered salineat 4° C. Fractions of about 15 ml were collected at a flow rate of about75 ml/h. In each case a major peak of material with absorbance at 280 nmeluted in fractions corresponding roughly to the elution volume range of1370 to 1635 ml. The fractions representing the absorbance peaks fromthe two column runs were combined into a single pool of 525 ml,containing about 2.3 g of SCF.

This material was sterilized by filtration using a Millipore Millipak 20membrane cartridge.

Alternatively, material from the C₄ column can be concentrated byultrafiltration and the buffer exchanged by diafiltration, prior tosterile filtration.

The isolated recombinant human SCF¹⁻¹⁶⁴ material is highly pure (>98% bySDS-PAGE with silver-staining) and is considered to be of pharmaceuticalgrade. Using the methods outlined in Example 2, it is found that thematerial has amino acid composition and amino acid sequence matchingthose expected from analysis of the SCF gene. The N-terminal amino acidsequence is Met-Glu-Gly-Ile . . . , i.e., the initiating Met residue isretained.

By procedures comparable to those outlined for human SCF¹⁻¹⁶⁴ expressedin E. coli, rat SCF¹⁻¹⁶⁴ (also present in insoluble form inside the cellafter fermention) can be recovered in a purified state with highbiological specific activity. Similarly, human SCF¹⁻¹⁸³ and rat SCF¹⁻¹⁹³can be recovered. The rat SCF¹⁻¹⁹³, during folding/oxidation, tends toform more variously oxidized species, and the unwanted species are moredifficult to remove chromatographically.

The rat SCF¹⁻¹⁹³ and human SCF¹⁻¹⁸³ are prone to proteolytic degradationduring the early stages of recovery, i.e., solubilization andfolding/oxidation. A primary site of proteolysis is located betweenresidues 160 and 170. The proteolysis can be minimized by appropriatemanipulation of conditions (e.g., SCF concentration; varying pH;inclusion of EDTA at 2-5 mM, or other protease inhibitors), and degradedforms to the extent that they are present can be removed by appropriatefractionation steps.

While the use of urea for solubilization, and during folding/oxidation,as outlined, is a preferred embodiment, other solubilizing agents suchas guanidine-HCl (e.g. 6 M during solubilization and 1.25 M duringfolding/oxidation) and sodium N-lauroyl sarcosine can be utilizedeffectively. Upon removal of the agents after folding/oxidation,purified SCFs, as determined by SDS-PAGE, can be recovered with the useof appropriate fractionation steps.

In addition, while the use of glutathione at 1 mM duringfolding/oxidation is a preferred embodiment, other conditions can beutilized with equal or nearly equal effectiveness. These include, forexample, the use in place of 1 mM glutathione of 2 mM glutathione plus0.2 mM oxidized glutathione, or 4 mM glutathione plus 0.4 mM oxidizedglutathione, or 1 mM 2-mercaptoethanol, or other thiol reagents also.

In addition to the chromatographic procedures described, otherprocedures which are useful in the recovery of SCFs in a purified activeform include hydrophobic interaction chromatography [e.g., the use ofphenyl-Sepharose (Pharmacia), applying the sample at neutral pH in thepresence of 1.7 M ammonium sulfate and eluting with a gradient ofdecreasing ammonium sulfate]; immobilized metal affinity chromatography[e.g., the use of chelating-Sepharose (Pharmacia) charged with Cu²⁺ ion,applying the sample at near neutral pH in the presence of 1 mM imidazoleand eluting with a gradient of increasing imidazole]; hydroxylapatitechromatography, [applying the sample at neutral pH in the presence of 1mM phosphate and eluting with a gradient of increasing phosphate]; andother procedures apparent to those skilled in the art.

Other forms of human SCF, corresponding to all or part of the openreading frame encoding by amino acids 1-248 in FIG. 42, or correspondingto the open reading frame encoded by alternatively spliced mRNAs thatmay exist (such as that represented by the cDNA sequence in FIG. 44),can also be expressed in E. coli and recovered in purified form byprocedures similar to those described in this Example, and by otherprocedures apparent to those skilled in the art.

The purification and for mulation of forms including the so-calledtransmembrane region referred to in Example 16 may involve theutilization of detergents, including non-ionic detergents, and lipids,including phospholipid-containing liposome structures.

B. SCF¹⁻¹⁶⁵

For the purification of human SCF¹⁻¹⁶⁵ expressed in E. coli, thefollowing information is relevant. After harvesting of cells expressingthe human SCF¹⁻¹⁶⁵, pharmaceutical grade human SCF¹⁻¹⁶⁵ was recovered byprocedures the same as those described for human SCF¹⁻¹⁶⁴ (above), butwith the following modifications. After cell lysis, the homogenate wasdiluted to a volume representing twice the volume of the original cellsuspension, with the inclusion of EDTA to 10 mM final concentration.Centrifugation was then done using a Sharples AS-16 centrifuge at 15,000rpm and flow rate of 0.5 L/min, to obtain a pellet fraction. This pelletfraction, without washing, was then subjected to the solubilization withurea, essentially as described for human SCF¹⁻¹⁶⁴ except that sodiumacetate was omitted, the mixture was titratated to pH 3 using HCl, theestimated SCF concentration was 3.2 mg/ml, and incubation was for 1-2 hat room temperature. All subsequent steps were at room temperature also.For refolding/reoxidation, the mixture was then diluted directly, by afactor of 3.2, such that the final conditions included the SCF at about1 mg/ml, 2.5 M urea, 60 mM NaCl, 1 mM glutathione, 50 mM Tris-HCl, withpH at 8.5. After stirring for 20-24 h, clarification was accomplished byfiltration through a Cuno Zeta Plus 30SP depth filtration device. A 19ft² filter was used per 100 L of mixture to be filtered. Flow rateduring filtration was about 2.9 L/min. For a 19 ft² filter, washing ofthe filter with 50 L of 20 mM Tris-HCl, pH 8.5 was done. The followingdescription applies to the handling of fractions derived from 100 L ofrefolding/reoxidation mixture. The 150 L of filtrate plus wash wasconcentrated to 50 L by ultrafiltration, and diafiltration against 300 Lof 20 mM Tris-HCl, pH 8.5 was then done. The diafiltered material wasthen diluted to 150 L by addition of the Tris buffer. pH was thenadjusted to 4.55 using 10% acetic acid, whereupon the mixture becameturbid. 2-24 h later, clarification was accomplished by depth filtrationusing a 19 ft² Cuno Zeta Plus 10SP filter, pre-washed with 0.1 M sodiumchloride, 50 mM sodium acetate, pH 4.5. After the filtration, the filterwas washed with 50 L of the same sodium chloride/sodium acetate buffer.The resulting filtrate plus wash (about 200 L) was applied to anS-Sepharose Fast Flow (Pharmacia) column (10 L bed volume; 30 cmdiameter) equilibrated with 50 mM sodium acetate, 100 mM sodiumchloride, pH 4.5. Flow rate was 1.4 L/min. After sample application, thecolumn was washed with 100 L of the column buffer, at a flow rate of 1.2L/min. Elution was carried out with a linear gradient from the startingcolumn buffer to 50 mM sodium acetate, 300 mM NaCl, pH 4.5 (200 L totalgradient volume), at flow rate of 0.65 L/min. The various formsdescribed for the S-Sepharose Fast Flow fractions obtained inpreparation of E. coli-derived human SCF¹⁻¹⁶⁴ above were present inessentially the same fashion, and pooling of fractions was based on thesame criteria as described above. The pooled material (about 25 g SCF inabout 20-25 L) was adjusted to pH 2.2 using 6 N HCl, and loaded onto aC4 column (1.2 L bed volume; 14 cm diameter; Vydac Proteins C₄, Cat. No.214TPB2030), at 100 ml/min. The column was next washed with 10 L of 25%ethanol, 12.5 mM HCl, and theneluted with a linear gradient fromthisbuffer to 75% ethanol, 12.5 mM HCl (25 L total gradient volume). Again,the various species present in the eluted fractions, and the pooling offractions, were essentially as described for the SCF¹⁻¹⁶⁴. The pool,containing about 16 g SCF¹⁻¹⁶⁵ correctly-oxidized monomer in a volume ofabout 9 ml, was diluted 6.25-fold, made 10 mM in sodium phosphate byaddition of 0.5 M sodium phosphate, pH 6.5, and titrated to pH 6.5 using1 N sodium hydroxide. The material was then applied at a flow rate of400 ml/min to a Q-Sepharose Fast Flow (Pharmacia) column (2 L bedvolume; 14 cm diamter) equilibrated with 10 mm sodium phosphate, pH 6.5.After washing the column with 20 L of 10 mM sodium phosphate, 25 mMsodium chloride, pH 6.5, elution was carried out with a linear gradientfrom the wash buffer to 10 mM sodium phosphate, 100 mM NaCl, pH 6.5.Fractions corresponding to the main absorbance (at 280 nm) peakrepresent the correctly-oxidized SCF¹⁻¹⁶⁵. These fractions were pooled;typically the pool contained about 12-15 g SCF¹⁻¹⁶⁵, in a volume ofabout 17-18 L. The SCF material was then concentrated by ultrafiltrationand other buffers optionally introduced by diafiltration, a preferredbuffer being 10 mM sodium acetate, 140 mM sodium chloride, pH 5.

C. SCF¹⁻²⁴⁸

The full length recombinant human stem cell factor (SCF¹⁻²⁴⁸) is formedin E. coli as inclusion bodies. After isolation of the inclusion bodies,treatment with 8M urea, 50 mm sodium acetate, 0.1 mM EDTA, pH 5.0 doesnot solubilize any SCF¹⁻²⁴⁸. This is in contrast to shorter SCFs whichsolubilize well in this buffer. To solubilize SCF¹⁻²⁴⁸, the urea-washedinclusion bodies are suspended in 50 mM Tris-HCl, 1 mM EDTA, 2% sodiumdeoxycholate (NaDOC), pH 8.5 at an approximate SCF¹⁻²⁴⁸ concentration of0.2 to 1.0 mg/mL. To this is added powdered dithiothreitol (DTT) to aconcentration of 20 mM. The mixture is stirred for 2.5 hours at roomtemperature. Unsolubilized debris is removed by centrifuing at 20,000×gfor 20 min. The supernatant contains all of the SCF¹⁻²⁴⁸ which runs as afuzzy 33,000 dalton band on a reducing SDS polyacrylamide gel. BothNaDOC, an anionic detergent, and DTT, a reducing agent are required forsolubilization.

Soluble oxidized SCF¹⁻²⁴⁸ can be prepared by diluting the solubilizationmixture supernatant with nine volumes of 50 mM Tris, 1 mM EDTA, 2% NaDOC(no pH adjustment). The pH of the diluted mixture is approximately 9.5.This mixture is stirred vigorously at room temperature for approximately40 hours. This mixture can be clarified by filtration through a 0.45μcellulose acetate membrane. The filtrate contains SCF¹⁻²⁴⁸ which runs asa 28,000 dalton band on a non-reducing SDS polyacrylamide gel. Underreducing conditions, the fuzzy 33,000 dalton band is visible. Thefiltrate also contains smaller but variable amounts of incompletelyoxidized SCF¹⁻²⁴⁸ and an apparent disulfide-linked dimer atapproximately 80,000 daltons on the gels. Upon removal of NaDOC bydiafiltration using a 10,000 dalton molecular weight cut-off membrane,the oxidized SCF¹⁻²⁴⁸ remains in solution.

SCF¹⁻²⁴⁸ was subsequently purified to 80-90% purity by a combination ofanion exchange, gel filtration, and cation exchange chromatography. Theprotein requires the presence of the non-ionic detergent, Triton X-100,to remain unaggregated. Material following anion exchange chromatographywas active in the UT-7 assay (Example 9B). The final material aftercation exchange chromatography showed no activity in the UT-7 assay. Itmay be that earlier samples contained some active proteolyzed SCF. TheSCF¹⁻²⁴⁸ diluted in detergent-free buffer for assay may be incapable ofinteraction with the SCF receptor because of aggregation.

EXAMPLE 11 Recombinant SCF from Mammalian Cells

A. Fermentation of CHO Cells Producing SCF

Recombinant Chinese hamster ovary (CHO) cells (strain CHO pDSRα2hSCF¹⁻¹⁶²) were grown on microcarriers in a 20 liter perfusion culturesystem for the production of human SCF¹⁻¹⁶². The fermentor system issimilar to that used for the culture of BRL 3A cells, Example 1B, exceptfor the following: The growth medium used for the culture of CHO cellswas a mixture of Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12nutrient mixture in a 1:1 proportion (GIBCO), supplemented with 2 mMglutamine, nonessential amino acids (to double the existingconcentration by using 1:100 dilution of Gibco #320-1140) and 5% fetalbovine serum. The harvest medium was identical except for the omissionof serum. The reactor was inoculated with 5.6×10⁹ CHO cells grown in two3-liter spinner flasks. The cells were allowed to grow to aconcentration of 4×10⁵ cells/ml. At this point 100 grams ofpresterilized cytodex-2 microcarriers (Pharmacia) were added to thereactor as a 3-liter suspension in phosphate buffered saline. The cellswere allowed to attach and grow on the microcarriers for four days.Growth medium was perfused through the reactor as needed based onglucose consumption. The glucose concentration was maintained atapproximately 2.0 g/L. After four days, the reactor was perfused withsix volumes of serum-free medium to remove most of the serum (proteinconcentration <50 μg/ml). The reactor was then operated batch-wise untilthe glucose concentration fell below 2 g/L. From this point onward, thereactor was operated at a continuous perfusion rate of approximately 20L/day. The pH of the culture was maintained at 6.9±0.3 by adjusting theCO₂ flow rate. The dissolved oxygen was maintained higher than 20% ofair saturation by supplementing with pure oxygen as necessary. Thetemperature was maintained at 37±0.5° C.

Approximately 450 liters of serum-free conditioned medium was generatedfrom the above system and was used as starting material for thepurification of recombinant human SCF¹⁻¹⁶².

Approximately 589 liters of serum-free conditioned medium was alsogenerated in similar fashion but using strain CHO pDSRα2 rSCF¹⁻¹⁶² andused as starting material for purification of rat SCF¹⁻¹⁶².

B. Purification of Recombinant Mammalian Expressed Rat SCF¹⁻¹⁶² andOther Recombinant Mammalian SCFs

All purification work was carried out at 4° C. unless indicatedotherwise.

1. Concentration and Diafiltration

Conditioned medium generated by serum-free growth of cell strain CHOpDSRα2 rat SCF¹⁻¹⁶² as performed in Section A above, was clarified byfiltration thru 0.45μ Sartocapsules (Sartorius). Several differentbatches (36 L, 101 L, 102 L, 200 L and 150 L) were separately subjectedto concentration and diafiltration/buffer exchange. To illustrate, thehandling of the 36 L batch was as follows. The filtered condition mediumwas concentrated to ˜500 ml using a Millipore Pellicon tangential flowultrafiltration apparatus with three 10,000 molecular weight cutoffcellulose acetate membrane cassettes (15 ft² total membrane area; pumprate ˜2,200 ml/min and filtration rate ˜750 ml/min).Diafiltration/buffer exchange in preparation for anion exchangechromatography was then accomplished by adding 1000 ml of 10 mMTris-HCl, pH 6.7-6.8 to the concentrate, reconcentrating to 500 ml usingthe tangential flow ultrafiltration apparatus, and repeating this 5additional times. The concentrated/diafiltered preparation was finallyrecovered in a volume of 1000 ml. The behavior of all conditioned mediumbatches subjected to the concentration and diafiltration/buffer exchangewas similar. Protein concentrations for the batches, determined by themethod of Bradford [Anal. Bioch. 72, 248-254 (1976)] with bovine serumalbumin as standard, were in the range 70-90 μg/ml. The total volume ofconditioned medium utilized for this preparation was about 589 L.

2. Q-Sepharose Fast Flow Anion Exchange Chromatography

The concentrated/diafiltered preparations from each of the fiveconditioned medium batches referred to above were combined (total volume5,000 ml). pH was adjusted to 6.75 by adding 1 M HCl. 2000 ml of 10 mMTris-HCl, pH 6.7 was used to bring conductivity to about 0.700 mmho. Thepreparation was applied to a Q-Sepharose Fast Flow anion exchange column(36×14 cm; Pharmacia Q-Sepharose Fast Flow resin) which had beenequilibrated with the 10 mM Tris-HCl, pH 6.7 buffer. After sampleapplication, the column was washed with 28,700 ml of the Tris buffer.Following this washing the column was washed with 23,000 ml of 5 mMacetic acid/1 mM glycine/6 M urea/20 μM CuSO₄ at about pH 4.5. Thecolumn was then washed with 10 mM Tris-HCl, 20 μm CuSO₄, pH 6.7 bufferto return to neutral pH and remove urea, and a salt gradient (0-700 mMNaCl in the 10 mM Tris-HCl, 20 μM CuSO₄, pH 6.7 buffer; 40 L totalvolume) was applied. Fractions of about 490 ml were collected at a flowrate of about 3,250 ml/h. The chromatogram is shown in FIG. 36. “MC/9cpm” refers to biological activity in the MC/9 assay; 5 μl from theindicated fractions was assayed. Eluates collected during the sampleapplication and washes are not shown in the Figure; no biologicalactivity was detected in these fractions.

3. Chromatography Using Silica-Bound Hydrocarbon Resin

Fractions 44-66 from the run shown in FIG. 36 were combined (11,200 ml)and EDTA was added to a final concentration of 1 mM. This material wasapplied at a flow rate of about 2000 ml/h to a C₄ column (Vydac ProteinsC₄; 7×8 cm) equilibrated with buffer A (10 mM Tris pH 6.7/20% ethanol).After sample application the column was washed with 1000 ml of buffer A.A linear gradient from buffer A to buffer B (10 mM Tris pH 6.7/94%ethanol) (total volume 6000 ml) was then applied, and fractions of 30-50ml were collected. Portions of the C₄ column starting sample, runthroughpool and wash pool in addition to 0.5 ml aliquots of the gradientfractions were dialyzed against phosphate-buffered saline in preparationfor biological assay. These various fractions were assayed by the MC/9assay (5 μl aliquots of the prepared gradient fractions; cpm in FIG.37). SDS-PAGE [Laemmli, Nature 227, 680-685 (1970); stacking gelscontained 4% (w/v) acrylamide and separating gels contained 12.5% (w/v)acrylamide) of aliquots of various fractions is shown in FIG. 38. Forthe gels shown, sample aliquots (100 μl) were dried under vacuum andthen redissolved using 20 μl sample treatment buffer (reducing, i.e.,with 2-mercaptoethanol) and boiled for 5 min prior to loading onto thegel. The numbered marks at the left of the Figure represent migrationpositions of molecular weight markers (reduced) as in FIG. 6. Thenumbered lanes represent the corresponding fractions collected duringapplication of the last part of the gradient. The gels weresilver-stained [Morrissey, Anal. Bioch. 117, 307-310 (1981)].

4. Q-Sepharose Fast Flow Anion Exchange Chromatography

Fractions 98-124 from the C₄ column shown in FIG. 37 were pooled (1050ml). The pool was diluted 1:1 with 10 mM Tris, pH 6.7 buffer to reduceethanol concentration. The diluted pool was then applied to aQ-Sepharose Fast Flow anion exchange column (3.2×3 cm, PharmaciaQ-Sepharose Fast Flow resin) which had been equilibratd with the 10 mMTris-HCl₁, pH 6.7 buffer. Flow rate was 463 ml/h. After sampleapplication the column was washed with 135 ml of column buffer andelution of bound material was carried out by washing with 10 mMTris-HCl, 350 mM NaCl, pH 6.7. The flow direction of the column wasreversed in order to minimize volume of eluted material, and 7.8 mlfractions were collected during elution.

5. Sephacryl S-200 HR Gel Filtration Chromatography

Fractions containing eluted protein from the salt wash of theQ-Sepharose Fast Flow anion exchange column were pooled (31 ml). 30 mlwas applied to a Sephacryl S-200 HR (Pharmacia) gel filtration column,(5×55.5 cm) equilibrated in phosphate-buffered saline. Fractions of 6.8ml were collected at a flow rate of 68 ml/hr. Fractions corresponding tothe peak of absorbance at 280 nm were pooled and represent the finalpurified material.

Table 15 shows a summary of the purification.

TABLE 15 Summary of Purification of Mammalian Expressed Rat SCF¹⁻¹⁶²Total Step Volume (ml) Protein (mg)* Conditioned medium (concentrated) 7,000 28,420    Q-Sepharose Fast Flow 11,200 974  C₄ resin  1,050 19Q-Sepharose Fast Flow    31 20 Sephacryl S-200 HR    82  19***Determined by the method of Bradford (supra, 1976). **Determined as47.3 mg by quantitative amino acid analysis using methodology similar tothat outlined in Example 2.

The N-terminal amino acid sequence of purified rat SCF¹⁻¹⁶² isapproximately half Gln-Glu-Ile . . . and half PyroGlu-Glu-Ile . . . , asdetermined by the methods outlined in Example 2. This result indicatesthat rat SCF¹⁻¹⁶² is the product of proteolytic processing/cleavagebetween the residues indicated as numbers (−1) (Thr) and (+1) (Gln) inFIG. 14C. Similarly, purified human SCF¹⁻¹⁶² from transfected CHO cellconditioned medium (below) has N-terminal amino acid sequenceGlu-Gly-Ile, indicating that it is the product of processing/cleavagebetween residues indicated as numbers (−1) (Thr) and (+1) (Glu) in FIG.15C.

Using the above-described protocol will yield purified human SCFprotein, either recombinant forms expressed in CHO cells or naturallyderived.

Additional purification methods that are of utility in the purificationof mammalian cell derived recombinant SCFs include those outlined inExamples 1 and 10, and other methods apparent to those skilled in theart.

Other forms of human SCF, corresponding to all or part of the openreading frame encoded by amino acids 1-248 shown in FIG. 42, orcorresponding to the open reading frame encoded by alternatively splicedmRNAs that may exist (such as that represented by the cDNA sequence inFIG. 44), can also be expressed in mammalian cells and recovered inpurified form by procedures similar to those decribed in this Example,and by other procedures apparent to those skilled in the art.

C. SDS-PAGE and Glycosidase Treatments

SDS-PAGE of pooled fractions from the Sephacryl S-200 HR gel filtrationcolumn is shown in FIG. 39; 2.5 μl of the pool was loaded (lane 1). Thelane was silver-stained. Molecular weight markers (lane 6) were asdescribed for FIG. 6. The different migrating material above andslightly below the M_(r) 31,000 marker position represents thebiologically active material; the apparent heterogeneity is largely dueto the heterogeneity in glycosylation.

To characterize the glycosylation purified material was treated with avariety of glycosidases, analyzed by SDS-PAGE (reducing conditions) andvisualized by silver-staining. Results are shown in FIG. 39. Lane 2,neuraminidase. Lane 3, neuraminidase and O-glycanase. Lane 4,neuraminidase, O-glycanase and N-glycanase. Lane 5, neuraminidase andN-glycanase. Lane 7, N-glycanase. Lane 8, N-glycanase without substrate.Lane 9, O-glycanase without substrate. Conditions were 10 mM3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane sulfonate (CHAPS),66.6 mM 2- mercaptoethanol, 0.04% (wt/vol) sodium azide, phosphatebuffered saline, for 30 min at 37° C., followed by incubation at half ofdescribed concentrations in presence of glycosidases for 18 h at 37° C.Neuraminidase (from Arthrobacter ureafaciens; supplied by Calbiochem)was used at 0.5 units/ml final concentration. O-Glycanase (Genzyme;endo-alpha-N-acetyl galactosaminidase) was used at 7.5 milliunits/ml.N-Glycanase (Genzyme; peptide: N-glycosidase F;peptide-N⁴[N-acetyl-beta-glucosaminyl]asparagine amidase) was used at 10units/ml.

Where appropriate, various control incubations were carried out. Theseincluded: incubation without glycosidases, to verify that results weredue to the glycosidase preparations added; incubation with glycosylatedproteins (e.g. glycosylated recombinant human erythropoietin) known tobe substrates for the glycosidases, to verify that the glycosidaseenzymes used were active; and incubation with glycosidases but nosubstrate, to judge where the glycosidase preparations were contributingto or obscuring the visualized gel bands (FIG. 39, lanes 8 and 9).

A number of conclusions can be drawn from the experiments describedabove. The various treatments with N-glycanase [which removes bothcomplex and high-mannose N-linked carbohydrate (Tarentino et al.,Biochemistry 24, 4665-4671 (1988)], neuraminidase (which removes sialicacid residues), and O-glycanase [which removes certain O-linkedcarbohydrates (Lambin et al., Biochem. Soc. Trans. 12, 599-600 (1984)],suggest that: both N-linked and O-linked carbohydrates are present; andsialic acid is present, with at least some of it being part of theO-linked moieties. The fact that treatment with N-glycanase can convertthe heterogeneous material apparent by SDS-PAGE to a faster-migratingform which is much more homogeneous indicates that all of the materialrepresents the same polypeptide, with the heterogeneity being causedmainly by heterogeneity in glycosylation.

While the results of this section apply to purified CHO cell-derived ratSCF¹⁻¹⁶², equivalent results of SDS-PAGE and glycosidase treatments areobtained for CHO cell-derived human SCF¹⁻¹⁶².

EXAMPLE 12 Preparation of Recombinant SCF PEG

A. Preparation of Recombinant SCF¹⁻¹⁶⁴ PEG

Rat SCF¹⁻¹⁶⁴, purified from a recombinant E. coli expression systemaccording to Examples 6A and 10, was used as starting material forpolyethylene glycol modification described below.

Methoxypolyethylene glycol-succinimidyl succinate (18.1 mg=3.63 umol;SS-MPEG=Sigma Chemical Co. no. M3152, approximate molecularweight=5,000) in 0.327 mL deionized water was added to 13.3 mg (0.727umol) recombinant rat SCF¹⁻¹⁶⁴ in 1.0 mL 138 mM sodium phosphate, 62 mMNaCl, 0.62 mM sodium acetate, pH 8.0. The resulting solution was shakengently (100 rpm) at room temperature for 30 minutes. A 1.0 mL aliquot ofthe final reaction mixture (10 mg protein) was then applied to aPharmacia Superdex 75 gel filtration column (1.6×50 cm) and eluted with100 mM sodium phosphate, pH 6.9, at a rate of 0.25 mL/min at roomtemperature. The first 10 mL of column effluent were discarded, and 1.0mL fractions were collected thereafter. The UV absorbance (280 nm) ofthe column effluent was monitored continuously and is shown in FIG. 40A.Fractions number 25 through 27 were combined and sterilized byultrafiltration through a 0.2μ polysulfone membrane (Gelman Sciences no.4454), and the resulting pool was designated PEG-25. Likewise, fractionsnumber 28 through 32 were combined, sterilized by ultrafiltration, anddesignated PEG-32. Pooled fraction PEG-25 contained 3.06 mg protein andpooled fraction PEG-32 contained 3.55 mg protein, as calculated fromA280 measurements using for calibration an absorbance of 0.66 for a 1.0mg/mL solution of unmodified rat SCF¹⁻¹⁶⁴. Unreacted rat SCF¹⁻¹⁶⁴,representing 11.8% of the total protein in the reaction mixture, waseluted in fractions number 34 to 37. Under similar chromatographicconditions, unmodified rat SCF¹⁻¹⁶⁴ was eluted as a major peak with aretention volume of 45.6 mL, FIG. 40B. Fractions number 77 to 80 in FIG.40A contained N-hydroxysuccinimide, a by-product of the reaction of ratSCF¹⁻¹⁶⁴ with SS-MPEG.

Potentially reactive amino groups in rat SCF¹⁻¹⁶⁴ include 12 lysineresidues and the alpha amino group of the N-terminal glutamine residue.Pooled fraction PEG-25 contained 9.3 mol of reactive amino groups permol of protein, as determined by spectroscopic titration withtrinitrobenzene sulfonic acid (TNBS) using the method described byHabeeb, Anal. Biochem. 14:328-336 (1966). Likewise, pooled fractionPEG-32 contained 10.4 mol and unmodified rat SCF¹⁻¹⁶⁴ contained 13.7 molof reactive amino groups per mol of protein, respectively. Thus, anaverage of 3.3 (13.7 minus 10.4) amino groups of rat SCF¹⁻¹⁶⁴ in pooledfraction PEG-32 were modified by reaction with SS-MPEG. Similarly, anaverage of 4.4 amino groups of rat SCF¹⁻¹⁶⁴ in pooled fraction PEG-25were modified. Human SCF (hSCF¹⁻¹⁶⁴) produced as in Example 10 was alsomodified using the procedures noted above. Specifically, 714 mg (38.5umol) hSCF¹⁻¹⁶⁴ were reacted with 962.5 mg (192.5 umol) SS-MPEG in 75 mLof 0.1 M sodium phosphate buffer, pH 8.0 for 30 minutes at roomtemperature. The reaction mixture was applied to a Sephacryl S-200HRcolumn (5×134 cm) and eluted with PBS (Gibco Dulbecco'sphosphate-buffered saline without CaCl₂ and MgCl₂) at a rate of 102mL/hr, and 14.3-mL fractions were collected. Fractions no. 39-53,analogous to the PEG-25 pool described above and in FIG. 40A, werepooled and found to contain a total of 354 mg of protein. In vivoactivity of this modified SCF in primates is presented in Example 8C.

B. Preparation of Recombinant SCF¹⁻¹⁶⁵PEG

Recombinant human SCF¹⁻¹⁶⁵ produced as in Example 10 was coupled tomethoxypolyethylene glycol (MW=6,000) by reacting 334 mg (18.0 μmol) ofrhuSCF¹⁶⁵ with 433 mg (72.2 μmol) of the N-hydroxysuccinimidyl ester ofcarboxymethyl-MPEG [prepared by procedures described by Veronese, F. M.,et al., J. Controlled Release, 10:145-154 (1989) in 33.4 ml of 0.1 Mbicine buffer, pH 8.0 for 1 hour at room temperature. The reactionmixture was diluted with 134 ml of water for injection (WFI), titratedto pH 4.0 with 0.5 N HCl, filtered through a 0.20μ cellulose acetatefilter (Nalgene no. 156-4020), and applied at a rate of 5.0 ml/min to a2.6×19.5 cm column of S-Sepharose FF (Pharmacia) which had beenpreviously equilibrated with 20 mM sodium acetate, pH 4.0 at roomtemperature. Effluent from the column was collected in 8.0-ml fractions(no. 1-18) during sample loading, and the ultraviolet absorbance (A₂₈₀)of the effluent was monitored continuously. The column was thensequentially washed with 200 ml of the equilibration buffer at 5.0ml/min (fractions no. 19-44), with 200 ml of 20 mM sodium acetate, 0.5 MNaCl, pH 4.0 at 8.0 ml/min (fractions no. 45-69), and finally with 200ml of 20 mM sodium acetate, 1.0 M NaCl, pH 4.0 at 8.0 ml/min (fractionsno. 70-94). Fractions (no. 28-31 and 55-62) containing MPEG-rhu-SCF¹⁻¹⁶⁵were combined and dialyzed by ultrafiltration (Amicon YM-10 membrane)against 10 mM sodium acetate, 140 mM NaCl, pH 5.0 to yield 284 mg offinal product in a volume of 105 ml. The resulting MPEG-rhu-SCF¹⁶⁵ wasshown to be free of unbound MPEG and other reaction by-products byanalytical size-exclusion HPLC [Toso-Haas TSK G3000 SWXL and G4000 SWXLcolumns (each 0.68×30 cm; 5 u) connected in tandem; 0.1 M sodiumphosphate, pH 6.9 at 1.0 ml/min at room temperature; UV absorbance (280nm) and refractive index detectors in series].

EXAMPLE 13 SCF Receptor Expression on Leukemic Blasts

Leukemic blasts were harvested from the peripheral blood of a patientwith a mixed lineage leukemia. The cells were purified by densitygradient centrifugation and adherence depletion. Human SCF¹⁻¹⁶⁴ wasiodinated according to the protocol in Example 7. The cells wereincubated with different concentrations of iodinated SCF as described[Broudy, Blood, 75 1622-1626 (1990)]. The results of the receptorbinding experiment are shown in FIG. 41. The receptor density estimatedis approximately 70,000 receptors/cell.

EXAMPLE 14 Rat SCF Activity on Early Lymphoid Precursors

The ability of recombinant rat SCF¹⁻¹⁶⁴ (rrSCF¹⁻¹⁶⁴), to actsynergistically with IL-7 to enhance lymphoid cell proliferation wasstudied in agar cultures of mouse bone marrow. In this assay, thecolonies formed with rrSCF 1-164 alone contained monocytes, neutrophils,and blast cells, while the colonies stimulated by IL-7 alone or incombination with rrSCF¹⁻¹⁶⁴ contained primarily pre-B cells. Pre-Bcells, characterized as B220⁺, sIg⁻, cμ⁺, were identified by FACSanalysis of pooled cells using fluorescence-labeled antibodies to theB220 antigen [Coffman, Immunol. Rev., 69, 5 (1982)] and to surface Ig(FITC-goat anti-K, Southern Biotechnology Assoc., Birmingham, Ala.); andby analysis of cytospin slides for cytoplasmic μ expression usingfluorescence-labeled antibodies (TRITC-goat anti-μ, SouthernBiotechnology Assoc.,). Recombinant human IL-7 (rhIL-7) was obtainedfrom Biosource International (Westlake Village, Calif.). When rrSCF¹⁻¹⁶⁴was added in combination with the pre-B cell growth factor IL-7, asynergistic increase in colony formation was observed (Table 16),indicating a stimulatory role of rrSCF 164 on early B cell progenitors.

TABLE 16 Stimulation of Pre-B Cell Colony Formation by rrSCF¹⁻¹⁶⁴ inCombination with hIL-7 Growth Factors Colony Number¹ Saline 0 rrSCF¹⁻¹⁶⁴200 ng 13 ± 2  100 ng 7 ± 4  50 ng 4 ± 2 rhIL-7 200 ng 21 ± 6  100 ng 18± 6   50 ng 13 ± 6   25 ng 4 ± 2 rhIL-7 200 ng + rrSCF¹⁻¹⁶⁴ 200 ng 60 ±0  100 ng + 200 ng 48 ± 8   50 ng + 200 ng 24 ± 10  25 ng + 200 ng 21 ±2  ¹Number of colonies per 5 × 10⁴ mouse bone marrow cells plated.

Each value is the mean of triplicate dishes±SD.

EXAMPLE 15 Identification of the Receptor for SCF

A. c-kit is the Receptor for SCF¹⁻¹⁶⁴

To test whether SCF¹⁻¹⁶⁴ is the ligand for c-kit, the cDNA for theentire murine c-kit [Qiu et al., EMBO J., 7, 1003-1011 (1988)] wasamplified using PCR from the SCF¹⁻¹⁶⁴ responsive mast cell line MC/9[Nabel et al., Nature, 291, 332-334 (1981)] with primers designed fromthe published sequence. The ligand binding and transmembrane domains ofhuman c-kit, encoded by amino acids 1-549 [Yarden et al., EMBO J., 6,3341-3351 (1987)], were cloned using similar techniques from the humanerythroleukemia cell line, HEL [Martin and Papayannopoulou, Science,216, 1233-1235 (1982)]. The c-kit cDNAs were inserted into the mammalianexpression vector V19.8 transfected into COS-1 cells, and membranefractions prepared for binding assays using either rat or human¹²⁵I-SCF¹⁻¹⁶⁴ according to the methods described in Sections B and Cbelow. Table 17 shows the data from a typical binding assay. There wasno detectable specific binding of ¹²⁵I human SCF¹⁻¹⁶⁴ to COS-1 cellstransfected with V19.8 alone. However, COS-1 cells expressing humanrecombinant c-kit ligand binding plus transmembrane domains (hckit-LT1)did bind ¹²⁵I-hSCF¹⁻¹⁶⁴ (Table 17). The addition of a 200 fold molarexcess of unlabelled human SCF¹⁻¹⁶⁴ reduced binding to backgroundlevels. Similarly, COS-1 cells transfected with the full length murinec-kit (mckit-L1) bound rat ¹²⁵I-SCF¹⁻¹⁶⁴. A small amount of rat¹²⁵I-SCF¹⁻¹⁶⁴ binding was detected in COS-1 cells transfectants withV19.8 alone, and has also been observed in untransfected cells (notshown), indicating that COS-1 cells express endogenous c-kit. Thisfinding is in accord with the broad cellular distribution of c-kitexpression. Rat ¹²⁵I-SCF¹⁻¹⁶⁴ binds similarly to both human and murinec-kit, while human ¹²⁵I-SCF¹⁻¹⁶⁴ bind with lower activity to murinec-kit (Table 17). This data is consistent with the pattern of SCF¹⁻¹⁶⁴cross-reactivity between species. Rat SCF¹⁻¹⁶⁴ induces proliferation ofhuman bone marrow with a specific activity similar to that of humanSCF¹⁻¹⁶⁴, while human SCF¹⁻¹⁶⁴ induced proliferation of murine mastcells occurs with a specific activity 800 fold less than the ratprotein.

In summary, these findings confirm that the phenotypic abnormalitiesexpressed by W or S1 mutant mice are the consequences of primary defectsin c-kit receptor/ligand interactions which are critical for thedevelopment of diverse cell types.

TABLE 17 SCF¹⁻¹⁶³ Binding to Recombinant c-kit Expressed in COS-1 CellsCPM Bound^(a) Plasmid Human SCF¹⁻¹⁶⁴ Rat SCF¹⁻¹⁶⁴ Transfected¹²⁵I-SCF^(b) ¹²⁵I-SCF+cold^(c) ¹²⁵I-SCF^(d) ¹²⁵I-SCF+cold^(e) V19.8 2,160 2,150  1,100   550 V19.8: 59,350 2,380 70,000 1,100 hckit-LT1V19.8:  9,500 1,100 52,700   600 mckit-L1 ^(a)The average of duplicatemeasurements is shown; the experiment has been independently performedwith similar results three times. ^(b)1.6 nM human ¹²⁵I-SCF¹⁻¹⁶⁴ ^(c)1.6nM human ¹²⁵I-SCF¹⁻¹⁶⁴+320 nM unlabelled human SCF¹⁻¹⁶⁴ ^(d)1.6 nM rat¹²⁵I-SCF¹⁻¹⁶⁴ ^(e)1.6 nM rat ¹²⁵I-SCF¹⁻¹⁶⁴+320 nM unlabelled ratSCF¹⁻¹⁶⁴

B. Recombinant c-kit Expression in COS-1 Cells

Human and murine c-kit cDNA clones were derived using PCR techniques[Saiki et al., Science, 239, 487-491 (1988)] from total RNA isolated byan acid phenol/chloroform extraction procedure [Chomczynsky and Sacchi,Anal. Biochem., 162, 156-159, (1987)] from the human erythroleukemiacell line HEL and MC/9 cells, respectively. Unique sequenceoligonucleotides were designed from the published human and murine c-kitsequences. First strand cDNA was synthesized from the total RNAaccording to the protocol provided with the enzyme, Mo-MLV reversetranscription (Bethesda Research Laboratories, Bethesda, Md.), usingc-kit antisense oligonucleotides as primers. Amplification ofoverlapping regions of the c-kit ligand binding and tyrosine kinasedomains was accomplished using appropriate pairs of c-kit primers. Theseregions were cloned into the mammalian expression vector V19.8 (FIG. 17)for expression in COS-1 cells. DNA sequencing of several clones revealedindependent mutations, presumably arising during PCR amplification, inevery clone. A clone free of these mutations was constructed byreassembly of mutation-free restriction fragments from separate clones.Some differences from the published sequence appeared in all or in abouthalf of the clones; these were concluded to be the actual sequencespresent in the cell lines used, and may represent allelic differencesfrom the published sequences. The following plasmids were constructed inV19.8: V19.8:mckit-LT1, the entire murine c-kit; and V19.8:hckit-L1,containing the ligand binding plus transmembrane region (amino acids1-549) of human c-kit.

The plasmids were transfected into COS-1 cells essentially as describedin Example 4.

C. ¹²⁵I-SCF¹⁻¹⁶⁴ Binding to COS-1 Cells Expressing Recombinant c-kit

Two days after transfection, the COS-1 cells were scraped from the dish,washed in PBS, and frozen until use. After thawing, the cells wereresuspended in 10 mM Tris-HCl, 1 mM MgCl₂ containing 1 mM PMSF, 100μg/ml aprotinin, 25 μg/ml leupeptin, 2 μg/ml pepstatin, and 200 μg/mlTLCK-HCl. The suspension was dispersed by pipetting up and down 5 times,incubated on ice for 15 minutes, and the cells were homogenized with15-20 strokes of a Dounce homogenizer. Sucrose (250 mM) was added to thehomogenate, and the nuclear fraction and residual undisrupted cells werepelleted by centrifugation at 500×g for 5 min. The supernatant wascentrifuged at 25,000 g for 30 min. at 4° C. to pellet the remainingcellular debris. Human and rat SCF¹⁻¹⁶⁴ were radioiodinated usingchloramine-T [Hunter and Greenwood, Nature, 194, 495-496 (1962)]. COS-1membrane fractions were incubated with either human or rat ¹²⁵I-SCF¹⁻¹⁶⁴(1.6 nM) with or without a 200 fold molar excess of unlabelled SCF¹⁻¹⁶⁴in binding buffer consisting of RPMI supplemented with 1% bovine serumalbumin and 50 mM HEPES (pH 7.4) for 1 h at 22° C. At the conclusion ofthe binding incubation, the membrane preparations were gently layeredonto 150 μl of phthalate oil and centrifuged for 20 minutes in a BeckmanMicrofuge 11 to separate membrane bound ¹²⁵I-SCF¹⁻¹⁶⁴ from free¹²⁵I-SCF¹⁻¹⁶⁴. The pellets were clipped off and membrane associated¹²⁵I-SCF¹⁻¹⁶⁴ was quantitated.

EXAMPLE 16 Isolation of a Human SCF cDNA

A. Construction of the HT-1080 cDNA Library

Total RNA was isolated from human fibrosarcoma cell line HT-1080 (ATCCCCL 121) by the acid guanidinium thiocyanate-phenol-chloroformextraction method [Chomczynski et al., Anal. Biochem. 162, 156 (1987)],and poly(A) RNA was recovered by using oligo(dT) spin column purchasedfrom Clontech. Double-stranded cDNA was prepared from 2 μg poly(A) RNAwith a BRL (Bethesda Research Laboratory) cDNA synthesis kit under theconditions recommended by the supplier. Approximately 100 ng of columnfractionated double-stranded cDNA with an average size of 2 kb wasligated to 300 ng SalI/NotI digested vector pSPORT 1 [D'Alessio et al.,Focus, 12, 47-50 (1990)] and transformed into DH5α (BRL, Bethesda, Md.)cells by electroporation [Dower et al., Nucl. Acids Res., 16, 6127-6145(1988)].

B. Screening of the cDNA Library

Approximately 2.2×10⁵ primary transformants were divided into 44 poolswith each containing ˜5000 individual clones. Plasmid DNA was preparedfrom each pool by the CTAB-DNA precipitation method as described (DelSal et al., Biotechniques, 7, 514-519 (1989)]. Two micrograms of eachplasmid DNA pool was digested with restriction enzyme NotI and separatedby gel electrophoresis. Linearized DNA was transferred onto GeneScreenPlus membrane (DuPont) and hybridized with ³²P-labeled PCR generatedhuman SCF cDNA (Example 3) under conditions previously described [Lin etal., Proc. Natl. Acad. Sci. USA, 82, 7580-7584 (1985)]. Three poolscontaining positive signal were identified from the hybridization. Thesepools of colonies were rescreened by the colony-hybridization procedure[Lin et al., Gene 44, 201-209 (1986)] until a single colony was obtainedfrom each pool. The cDNA sizes of these three isolated clones arebetween 5.0 to 5.4 kb. Restriction enzyme digestions and nucleotidesequence determination at the 5′ end indicate that two out of the threeclones are identical (10-1a and 21-7a). They both contain the codingregion and approximately 200 bp of 5′ untranslated region (5′UTR). Thethird clone (26-1a) is roughly 400 bp shorter at the 5′ end than theother two clones. The sequence of this human SCF cDNA is shown in FIG.42. Of particular note is the hydrophobic transmembrane domain sequencestarting in the region of amino acids 186-190 and ending at amino acid212.

C. Construction of pDSRα2 hSCF¹⁻²⁴⁸

pDSRα2 hSCF¹⁻²⁴⁸ was generated using plasmids 10-1a (as described inExample 16B) and pGEM3 hSCF¹⁻¹⁶⁴ as follows: The HindIII insert frompGEM3 hSCF¹⁻¹⁶⁴ was transferred to M13mp18. The nucleotides immediatelyupstream of the ATG initiation codon were changed by site directedmutagenesis from tttccttATG (SEQ ID NO.:102) to gccgccgccATG (SEQ IDNO.:163) using the antisense oligonucleotide

5′-TCT TCT TCA TGG CGG CGG CAA GCT T 3′  (SEQ ID NO.:104)

and the oligonucleotide-directed in vitro mutagenesis system kit andprotocols from Amersham Corp. to generate M13mp18 hSCF^(K1-164). ThisDNA was digested with HindIII and inserted into pDSRα2 which had beendigested with HindIII. This clone is designated pDSRα2 hSCF^(K1-164).DNA from pDSRα2 hSCF^(K1-164) was digested with XbaI and the DNA madeblunt ended by the addition of Klenow enzyme and four dNTPs. Followingtermination of this reaction the DNA was further digested with theenzyme SpeI. Clone 10-1a was digested with DraI to generate a blunt end3′ to the open reading frame in the insert and with SpeI which cuts atthe same site within the gene in both pDSRα2 hSCF^(K1-164) and 10-1a.These DNAs were ligated together to generate pDSRα2 hSCF^(K1-248).

D. Transfection and immunoprecipitation of COS cells with pDSRα2hSCF¹⁻²⁴⁸ DNA.

COS-7 (ATCC CRL 1651) cells were transfected with DNA constructed asdescribed above. 4×10⁶ cells in 0.8 ml DMEM+5% FBS were electroporatedat 1600 V with either 10 μg pDSRα2 hSCF^(K1-248) DNA or 10 μg pDSRα2vector DNA (vector control). Following electroporation, cells werereplated into two 60-mm dishes. After 24 hrs, the medium was replacedwith fresh complete medium.

72 hrs after transfection, each dish was labelled with ³⁵S-mediumaccording to a modification of the protocol of Yarden et al. (PNAS 87,2569-2573, 1990). Cells were washed once with PBS and then incubatedwith methionine-free, cysteine-free DMEM (met⁻cys⁻DMEM) for 30 min. Themedium was removed and 1 ml met⁻cys⁻DMEM containing 100 μCi/mlTran³⁵S-Label (ICN) was added to each dish. Cells were incubated at 37°C. for 8 hr. The medium was harvested, clarified by centrifugation toremove cell debris and frozen at −20° C.

Aliquots of labelled conditioned medium of COS/pDSRα2 hSCF^(K1-248) andCOS/pDSRα2 vector control were immunoprecipitated along with mediumsamples of ³⁵S-labelled CHO/pDSRα2 hSCF¹⁻¹⁶⁴ clone 17 cells (see Example5) according to a modification of the protocol of Yarden et al. (EMBO,J., 6, 3341-3351, 1987). One ml of each sample of conditioned medium wastreated with 10 μl of pre-immune rabbit serum (#1379 P.I.). Samples wereincubated for 5 h. at 4° C. One hundred microliters of a 10% suspensionof Staphylococcus aureus (Pansorbin, Calbiochem.) in 0.15 M NaCl, 20 mMTris pH 7.5, 0.2% Triton X-100 was added to each tube. Samples wereincubated for an additional one hour at 4° C. Immune complexes werepelleted by centrifugation at 13,000×g for 5 min. Supernatants weretransferred to new tubes and incubated with 5 μl rabbit polyclonalantiserum (#1381 TB4), purified as in Example 11, against CHO derivedhSCF¹⁻¹⁶² overnight at 4° C. 100 μl Pansorbin was added for 1 h. andimmune complexes were pelleted as before. Pellets were washed 1× withlysis buffer (0.5% Na-deoxycholate, 0.5% NP-40, 50 mM NaCl, 25 mM TrispH 8), 3× with wash buffer (0.5 M NaCl, 20 mM Tris pH 7.5, 0.2% TritonX-100), and 1× with 20 mM Tris pH 7.5. Pellets were resuspended in 50 μl10 mM Tris pH 7.5, 0.1% SDS, 0.1 M β-mercaptoethanol. SCF protein waseluted by boiling for 5 min. Samples were centrifuged at 13,000×g for 5min. and supernatants were recovered.

Treatment with glycosidases was accomplished as follows: threemicroliters of 75 mM CHAPS containing 1.6 mU O-glycanase, 0.5 UN-glycanase, and 0.02 U neuraminidase was added to 25 μl of immunecomplex samples and incubated for 3 hr. at 37° C. An equal volume of2×PAGE sample buffer was added and samples were boiled for 3 min.Digested and undigested samples were electrophoresed on a 15%SDS-polyacrylamide reducing gel overnight at 8 mA. The gel was fixed inmethanol-acetic acid, treated with Enlightening enhancer (NEN) for 30min., dried, and exposed to Kodak XAR-5 film at −70°.

FIG. 43 shows the autoradiograph of the results. Lanes 1 and 2 aresamples from control COS/pDSRα2 cultures, lanes 3 and 4 fromCOS/pSRα2hSCF¹⁻²⁴⁸, lanes 5 and 6 from CHO/pDSRα2 hSCF¹⁻¹⁶⁴. Lanes 1, 3,and 5 are undigested immune precipitates; lanes 2, 4, and 6 have beendigested with glycanases as described above. The positions of themolecular weight markers are shown on the left. Processing of the SCF inCOS transfected with pDSRα2 hSCF¹⁻²⁴⁸ closely resembles that ofhSCF¹⁻¹⁶⁴ secreted from CHO transfected with pDSRα2 hSCF¹⁻¹⁶⁴, (Example11). This strongly suggests that the natural proteolytic processing sitereleasing SCF from the cell is in the vicinity of amino acid 164.

EXAMPLE 17 Quaternary Structure Analysis of Human SCF

Upon calibration of the gel filtration column (ACA 54) described inExample 1 for purification of SCF from BRL cell medium with molecularweight standards, and upon elution of purified SCF from other calibratedgel filtration columns, it is evident that SCF purified from BRL cellmedium behaves with an apparent molecular weight of approximately70,000-90,000 relative to the molecular weight standards. In contrast,the apparent molecular weight by SDS-PAGE is approximately28,000-35,000. While it is recognized that glycosylated proteins maybehave anomalously in such analyses, the results suggest that theBRL-derived rat SCF may exist as non-covalently associated dimer undernon-denaturing conditions. Similar results apply for recombinant SCFforms (e.g. rat and human SCF¹⁻¹⁶⁴ derived from E. coli, rat and humanSCF¹⁻¹⁶² derived from CHO cells) in that the molecular size estimated bygel filtration under non-denaturing conditions is roughly twice thatestimated by gel filtration under denaturing conditions (i.e., presenceof SDS), or by SDS-PAGE, in each particular case. Furthermoresedimentation velocity analysis, which provides an accuratedetermination of molecular weight in solution, gives a value of about36,000 for molecular weight of E. coli-derived recombinant humanSCF¹⁻¹⁶⁴. This value is again approximately twice that seen by SDS-PAGE(˜18,000-19,000). Therefore, while it is recognized that there may bemultiple oligomeric states (including the monomeric state), it appearsthat the dimeric state predominates under some circumstances insolution. CHO cell-derived human SCF¹⁻¹⁶² has a molecular weight ofabout 53,000 by sedimentation equilibrium analysis; this indicates thatit is dimeric also, and that it is about 30% carbohydrate by weight.

EXAMPLE 18 Isolation of Human SCF cDNA Clones from the 5637 Cell Line

A. Construction of the 5637 cDNA Library

Total RNA was isolated from human bladder carcinoma cell line 5637 (ATCCHTB-9) by the acid guanidinium thiocyanate-phenol-chloroform extractionmethod [Chomczynski et al., Anal. Biochem, 162, 156 (1987)], and poly(A)RNA was recovered by using an oligo(dT) spin column purchased fromClontech. Double-stranded cDNA was prepared from 2 μg poly(A) RNA with aBRL cDNA synthesis kit under the conditions recommended by the supplier.Approximately 80 ng of column fractionated double-stranded cDNA with anaverage size of 2 kb was ligated to 300 ng SalI/NotI digested vectorpSPORT 1 [D'Alessio et al., Focus, 12, 47-50 (1990)] and transformedinto DH5α cells by electroporation [Dower et al., Nucl. Acids Res., 16,6127-6145 (1988)].

B. Screening of the cDNA Library

Approximately 1.5×10⁵ primary tranformants were divided into 30 poolswith each containing approximately 5000 individual clones. Plasmid DNAwas prepared from each pool by the CTAB-DNA precipitation method asdescribed (Del Sal et al., Biotechniques, 7, 514-519 (1989)). Twomicrograms of each plasmid DNA pool was digested with restriction enzymeNotI and separated by gel electrophoresis. Linearized DNA wastransferred to GeneScreen Plus membrane (DuPont) and hybridized with³²P-labeled full length human SCF cDNA isolated from HT1080 cell line(Example 16) under the conditions previously described [Lin et al.,Proc. Natl. Acad. Sci. USA, 82, 7580-7584 (1985)]. Seven poolscontaining positive signal were identified from the hybridization. Thepools of colonies were rescreened with ³²P-labeled PCR generated humanSCF cDNA (Example 3) by the colony hybridization procedure [Lin et al.,Gene, 44, 201-209 (1986)] until a single colony was obtained from fourof the pools. The insert sizes of four isolated clones are approximately5.3 kb. Restriction enzyme digestions and nucleotide sequence analysisof the 5′-ends of the clones indicate that the four clones areidentical. The sequence of this human cDNA is shown in FIG. 44. The cDNAof FIG. 44 codes for a polypeptide in which amino acids 149-177 of thesequences in FIG. 42 are replaced by a single Gly residue.

EXAMPLE 19 SCF Enhancement of Survival After Lethal Irradiation

A. SCF in vivo activity on Survival After Lethal Irradiation.

The effect of SCF on survival of mice after lethal irradiation wastested. Mice used were 10 to 12 week-old female Balb/c. Groups of 5 micewere used in all experiments and the mice were matched for body weightwithin each experiment. Mice were irradiated at 850 rad or 950 rad in asingle dose. Mice were injected with factors alone or factors plusnormal Balb/c bone marrow cells. In the first case, mice were injectedintravenously 24 hrs. after irradiation with rat PEG-SCF¹⁻¹⁶⁴ (20μg/kg), purified from E. coli and modified by the addition ofpolyethylene glycol as in Example 12, or with saline for controlanimals. For the transplant model, mice were injected i.v. with variouscell doses of normal Balb/c bone marrow 4 hours after irradiation.′Treatment with rat PEG-SCF¹⁻¹⁶⁴ was performed by adding 200 μg/kg of ratPEG-SCF¹⁻¹⁶⁴ to the cell suspension 1 hour prior to injection and givenas a single i.v. injection of factor plus cells.

After irradiation at 850 rads, mice were injected with rat PEG-SCF¹⁻¹⁶⁴or saline. The results are shown in FIG. 45. Injection of ratPEG-SCF¹⁻¹⁶⁴ significantly enhanced the survival time of mice comparedto control animals (P<0.0001). Mice injected with saline survived anaverage of 7.7 days, while rat PEG-SCF¹⁻¹⁶⁴ treated mice survived anaverage of 9.4 days (FIG. 45). The results presented in FIG. 45represent the compilation of 4 separate experiments with 30 mice in eachtreatment group.

The increased survival of mice treated with rat PEG-SCF¹⁻¹⁶⁴ suggests aneffect of SCF on the bone marrow cells of the irradiated animals.Preliminary studies of the hematological parameters of these animalsshow slight increases in platelet levels compared to control animals at5 days post irradiation, however at 7 days post irradiation the plateletlevels are not significantly different to control animals. Nodifferences in RBC or WBC levels or bone marrow cellularity have beendetected.

B. Survival of Transplanted Mice Treated with SCF

Doses of 10% femur of normal Balb/c bone marrow cells transplanted intomice irradiated at 850 rad can rescue 90% or greater of animals (datanot presented). Therefore a dose of irradiation of 850 rad was used witha transplant dose of 5% femur to study the effects of rat PEG-SCF¹⁻¹⁶⁴on survival. At this cell dose it was expected that a large percentageof mice not receiving SCF would not survive; if rat PEG-SCF¹⁻¹⁶⁴ couldstimulate the transplanted cells there might be an increase in survival.As shown in FIG. 46, approximately 30% of control mice survived past 8days post irradiation. Treatment with rat PEG-SCF¹⁻¹⁶⁴ resulted in adramatic increase of survival with greater than 95% of these micesurviving out to at least 30 days (FIG. 46). The results presented inFIG. 46 represent the compilation of results from 4 separate experimentsrepresenting 20 mice in both the control and rat PEG-SCF¹⁻¹⁶⁴ treatedmice. At higher doses of irradiation, treatment of mice with ratPEG-SCF¹⁻¹⁶⁴ in conjunction with marrow transplant also resulted inincreased survival (FIG. 47). Control mice irradiated at 950 rads andtransplanted with 10% of a femur were dead by day 8, while approximately40% of mice treated with rat PEG-SCF¹⁻¹⁶⁴ survived 20 days or longer.20% of control mice transplanted with 20% of a femur survived past 20days while 80% of rSCF treated animals survived (FIG. 47).

C. Radioprotective Effects of SCF on Lethally Irradiated Mice Without aBone Marrow Transplant.

The effects of SCF administration prior to irradiation were compared tothe effects of SCF administration post-irradiation.

Female BDF1 mice (Charles River Laboratories, were used. All mice werebetween 7 and 8 weeks old and averaged 20-24 g each. Irradiationconsisted of a lethal split dose of 575 RADS each (total 1150 RADS)delivered 4 hours apart from a Gamma Cell to 40 duel cobalt source,(Atomic Energy Of Canada Limited).

In the experiment shown in FIG. 19-1, the ability of SCF, administeredprior to irradiation, to save mice from an otherwise lethal exposure wastested. Rat SCF, purified from E. coli as in Example 10 and modified bythe addition of polyethylene glycol as in Example 12, was administeredto two groups of mice (n=30), either intra-peritoneally or intravenouslyat a dose of 100 μg/kg. Control animals received excipient only whichconsisted of phosphate-buffered saline, 0.1% fetal bovine serum. Thetimes of administration were t=−20 hours and t=−2 hours to theirradiation event (t=0). The survival of the animals was monitoreddaily. The results are shown in FIG. 48. Both routes of administrationof rat SCF-PEG enhanced survival of the irradiated mice. At 30 days postirradiation, 100% of the animals treated with SCF were alive, whereasonly 35% of the animals in the control group were alive. Since similarexperiments, outlined in Example 19 A where SCF was administeredpost-irradiation only, yielded different results, the two modes ofadministration were compared directly in a single experiment. Theexperiment was performed as described above for FIG. 49 except thegroups were as follows (irradiation was at t=0): group 1, control; group2, rat SCF-PEG administered at t=−20 hours and t=−2 hours; group 3, ratSCF-PEG administered at t=−20 hours, t=−2 hours, and t=+4 hours; andgroup 4, rat SCF-PEG administered at t=+4 hours only. Both groupsreceiving rat SCF-PEG prior to irradiation survived at 95-100% at day 14(groups 2 and 3). In accordance with the experiment described in Example19 A, the animals receiving rat SCF-PEG post irradiation only did notsurvive the irradiation event, although they survived longer thancontrols.

These experiments demonstrate the utility of SCF administration toprotect against the lethal effects of irradiation. These protectiveeffects are most effective when SCF is administered prior to theirradiation event as well as after. This aspect of in vivo activity ofSCF can be utilized in dose intensification regimes in anti-neoplasticradiotherapy.

EXAMPLE 20 Production of Monoclonal Antibodies Against SCF

8-week old female BALB/c mice (Charles River, Wilmington, Mass.) wereinjected subcutaneously with 20 μg of human SCF¹⁻¹⁶⁴ expressed from E.coli in complete Freund's adjuvant (H37-Ra; Difco Laboratories, Detroit,Mich.). Booster immunizations of 50 μg of the same antigen in IncompleteFreund's adjuvant were subsequently administered on days 14, 38 and 57.Three days after the last injection, 2 mice were sacrificed and theirspleen cells fused with the sp 2/0 myeloma line according to theprocedures described by Nowinski et al., [Virology 93, 111-116 (1979)].

The media used for cell culture of sp 2/0 and hybridoma was Dulbecco'sModified Eagle's Medium (DMEM), (Gibco, Chagrin Falls, Ohio)supplemented with 20% heat inactivated fetal bovine serum (Phibro Chem.,Fort Lee, N.J.), 110 mg/ml sodium pyruvate, 100 U/ml penicillin and 100mcg/ml streptomycin (Gibco). After cell fusion hybrids were selected inHAT medium, the above medium containing 10⁻⁴M hypoxanthine, 4×10⁻⁷Maminopterin and 1.6×10⁻⁵M thymidine, for two weeks, then cultured inmedia containing hypoxanthine and thymidine for two weeks.

Hybridomas were screened as follows: Polystyrene wells (Costar,Cambridge, Mass.) were sensitized with 0.25 μg of human SCF¹⁻¹⁶⁴ (E.coli) in 50 μl of 50 mM bicarbonate buffer pH 9.2 for two hours at roomtemperature, then overnight at 4° C. Plates were then blocked with 5%BSA in PBS for 30 minutes at room temperature, then incubated withhybridoma culture supernatant for one hour at 37° C. The solution wasdecanted and the bound antibodies incubated with a 1:500 dilution ofGoat-anti-mouse IgG conjugated with Horse Radish Peroxidase (BoehringerMannheim Biochemicals, Indianapolis, Ind.) for one hour at 37° C. Theplates were washed with wash solution (KPL, Gaithersburg, Md.) thendeveloped with mixture of H₂O₂ and ABTS (KPL). Colorimetry was conductedat 405 nm.

Hybridoma cell cultures secreting antibody specific for human SCF¹⁻¹⁶⁴(E. coli) were tested by ELISA, same as hybridoma screening procedures,for crossreactivities to human SCF¹⁻¹⁶² (CHO). Hybridomas were subclonedby limiting dilution method. 55 wells of hybridoma supernatant testedstrongly positive to human SCF¹⁻¹⁶⁴ (E. coli); 9 of them crossreacted tohuman SCF¹⁻¹⁶² (CHO).

Several hybridoma cells have been cloned as follows:

Monoclone IgG Isotype Reactivity to human SCF¹⁻¹⁶² (CHO) 4G12-13 IgG1 No6C9A IgG1 No 8H7A 1gG1 Yes

Hybridomas 4G12-13 and 8H7A were deposited with the ATCC on Sep. 26,1990.

EXAMPLE 21 Synergistic Effect of SCF and Other Growth Factors

A. Synergistic Effect of SCF and G-CSF in Rodents

Lewis rats, male, weighing approximately 225 gms, were injectedintravenously via the dorsal vein of the penis with eitherpolyethylenesporeglycol-modified ratSCF-PEG (Examples 10 and 12),recombinant human G-CSF, a combination of both growth factors, or withcarrier consisting of 1% normal rat serum in sterile saline.Quantitative peripheral blood and bone marrow differentials wereperformed at various timepoints as previously described [Hulse, ActaHaematol. 31:50 (1964); Chervenick et al., Am. J. Physiol. 215: 353(1968)]. Histologic examination of the spleen was performed withBouin's-fixed paraffin-embedded sections stained withhematoxylin-and-eosin as well as by the Giemsa method. The numbers ofnormoblasts, megakaryocytes, and mast cells per 400× or 1000× high powerfield (HPF) in the spleen was quantitated by counting the number of eachcell type in randomly selected fields of the red pulp. Increases incirculating numbers of neutrophils over extended time periods were whenso stated calculated by planimetry as previously described. [Ulich etal., Blood 75:48 (1990)]. Data is expressed as the mean plus-or-minusone standard deviation and statistical analysis is by the unpairedt-test.

A single coinjection of ratSCF-PEG (25 ug/rat) plus G-CSF (25 ug/rat)causes an increase in circulating neutrophils that is approximatelyadditive (FIG. 50 CSF) as compared to ratSCF-PEG alone (25 ug/rat) orG-CSF alone (25 ug/rat) as measured by planimetry over a 35 hour timeperiod. The kinetics of ratSCF-PEG plus G-CSF-induced peripheralneutrophilia reflect the combined effect of the differing kinetics ofratSCF-induced neutrophilia peaking at 6 hours and G-CSF-inducedneutrophilia peaking at 12 hours (FIG. 50). The bone marrow at 6 hoursafter a single coinjection of ratSCF-PEG plus G-CSF (Table 18) shows agreater than additive decrease in mature marrow neutrophils(9.94±0.3×10⁶ PMN/humerus in carrier control rats vs. 2.11±0.3×10⁶PMN/humerus in ratSCF-PEG plus G-CSF-treated rats, 79% decrease) ascompared to ratSCF-PEG alone-treated rats (7.55±0.2×10⁶ PMN/humerus, 24%decrease) or G-CSF alone-treated rats (5.55±0.5×10⁶ PMN/humerus, 44%decrease). A significant increase in myeloblasts and promyelocytes wasseen in ratSCF-PEG, G-CSF-, and ratSCF-PEG plus G-CSF-treated rats at 6hours as compared to carrier controls (Table 18), but no significantincrease in any form of immature myeloid cells is noted in ratSCF-PEGplus G-CSF-treated rats as compared to ratSCF-PEG alone- or G-CSFalone-treated rats. A significant increase in myeloblasts is noted at 24hours, however, in the ratSCF-PEG plus G-CSF group as compared to eitherratSCF-PEG, G-CSF, or carrier alone (p<0.01, Table 19).

Daily coinjection of ratSCF-PEG (25 ug/rat) plus G-CSF (25 ug/rat) forone week causes a highly synergistic increase in circulating neutrophils(FIG. 51) as compared to ratSCF-PEG alone (25 ug/rat) or G-CSF alone (25ug/rat). A marked linear increase rise in the number of circulatingneutrophils occurs between day 4 and 6 after the coinjection ofratSCF-PEG plus G-CSF to 41.4±1.2×10³ PMN/mm³ at 24 hours after the lastinjection of the week as compared to 10.6±3.6×10³ PMN/mm³ in G-CSFtreated rats and 2.4±1.3×10³ PMN/mm³ in ratSCF-PEG alone treated rats(FIG. 51). A more detailed kinetic study of ratSCF-PEG plusG-CSF-induced neutrophilia after the last injection of the week showedthat the peak of circulating neutrophils occurs at 12 hours and reachesa level of 69.2±2.5×10³ PMN/mm³ as compared to 25.3±0.3×10³ PMN/mm³ inG-CSF-treated rats and 5.6±3.4×10³ in ratSCF-PEG-treated rats (FIG. 52).The neutrophils of ratSCF-PEG plus G-CSF-treated rats were extremelyhypersegmented (FIG. 52). In addition to the overwhelming increase inmature neutrophils in the circulation, an increase in immature myeloidforms was noted as well as the appearance of immature monocytoid forms,rare macrophage-like cells that contained vacuoles and ingestederythroid or lymphoid cells, rare basophils, rare mononuclearpromegakaryocytic forms and occasional late normoblasts in peripheralblood smears. As many as 3% of the nucleated circulating blood cellswere normoblasts in some of the peripheral blood smears of ratSCF-PEGplus G-CSF-treated rats after daily treatment for one week.

Two of the four rats in the ratSCF-PEG plus G-CSF-treated group died(one on the fifth day and one on the sixth day of the experiment), oneof the surviving rats appeared ill on the day of sacrifice (the seventhday), and both of the surviving rats were thrombocytopenic. None of therats in the ratSCF-PEG alone, G-CSF alone, or carrier control groupsshowed any evidence of morbidity or were thrombocytopenic.

The bone marrow at 24 hours after the daily coinjection of ratSCF-PEGplus G-CSF for one week demonstrated a synergistic increase in matureneutrophils form 10.6±0.6×10⁶ PMN/humerus in carrier controls,14.5±1.0×10⁶ PMN/humerus in ratSCF-PEG alone-treated rats, and28.5±2.1×10⁶ PMN/humerus in G-CSF alone-treated rats (Table 20). Theneutrophils in the marrow are generally hypersegmented and are oftenhypergranulated due to an increase in primary azurophilic granules.

The spleens of ratSCF-PEG plus G-CSF-treated rats were much larger andhistologic examination showed increased myelopoiesis, erythropoiesis,and megakaryocytopoiesis as compared to the spleens of control or singlefactor-treated rats. The spleens of ratSCF-PEG plus G-CSF-treated ratsshowed atrophy of the white pulp concomitant with a tremendous expansionof the red pulp which was replaced by nearly confluent extramedullaryhematopoiesis. The number of granulocytic precursors (myeloblasts tometamyelocytes) was readily seen by scanning histologic sections of thespleen to be markedly increased in the ratSCF-PEG plus G-CSF group ascompared to all other groups. Interestingly, the number of normoblastsin the spleen was also increased in the ratSCF-PEG plus G-CSF group(4.1±5.8 in the ratSCF-PEG alone group, 0±0 in the G-CSF alone group,and 36.4±26.1 in the ratSCF-PEG plus G-CSF group; 18 1,000×HPF/spleen/rat; p<0.0001 comparing ratSCF-PEG plus G-CSF vs. ratSCF-PEGalone). The mumber of megakaryocytes in the spleen was alsosignificantly increased in the ratSCF-PEG plus G-CSF group (1.8±1.5 inthe ratSCF-PEG alone group, 2.0±1.1 in the G-CSF alone group, and5.2±3.1 in the ratSCF-PEG plus G-CSF group; 12 400× HPF/spleen/rat;p<0.0001 comparing ratSCF-PEG plus G-CSF to either ratSCF-PEG or G-CSFalone).

These results demonstrate that the in vivo combination of ratSCF-PEG andG-CSF causes a synergistic myeloid hyperplasia in the bone marrow andspleen and a synergistic increase in circulating neutrophils. Thesynergism between a single dose of ratSCF-PEG and G-CSF becomes mostdramatically apparent as a rapidly increasing number of circulatingneutrophils between 4 and 6 hours after commencement of administrationof growth factors. Daily coinjection plus G-CSF for one week causes ahighly synergistic increase in circulating neutrophils as compared toratSCF-PEG alone or G-CSF alone.

B. Synergistic Effect of SCF and Other Growth Factors in Canines.

Though single factors such as G-CSF have been shown to have importanteffects on hematopoietic recovery, the combination of SCF with G-CSF hasa dramatic hematologic response. In the first set of experiments, 3normal dogs were treated with recombinant canine SCF alone at 200μg/kg/day subcutaneously or by continuous intravenous infusion. Theseanimals responded with an increase in the white blood cell count to30-50,000/mm³, from a baseline of 10-15,000 mm³ by day 8-12. Whenanother group of normal dogs were treated for 28 days with recombinantcanine SCF (200 μg/kg/day SCF and G-CSF (10 μg/kg/day SC), the whiteblood cell count increased from a normal range of 10-11,000/mm³ to200-240,000 cells/mm³ by day 17-21. This demonstrates that the effectsof SCF are dramatically enhanced in combination with other hematopoieticgrowth factors. Similarly, in vitro data show that SCF in combinationwith EPO dramatically enhances BFU-E growth (number and size, seeExample 9), again demonstrating that combinations of hematopoieticgrowth factors are more effective in eliciting a hematopoietic responseand/or may allow for lower doses of other factors to elicit the sameresponse.

EXAMPLE 22 The Use of SCF in Hematopoietic Transplantation

A. The Effects of SCF on Amplification of Bone Marrow and PeripheralBlood Hematopoietic Progenitors

The effects of SCF administration on circulating hematopoieticprogenitors in normal baboons was studied. The experimental design wasidentical to that described in Example 8C. Briefly, normal baboons wereadministered 200 μg/kg/day human SCF¹⁻¹⁶⁴, produced in E. coli as inExample 10 and modified by the addition of polyethylene glycol as inExample 12, as a continuous intravenous infusion. At various times bonemarrow and peripheral blood was harvested and cultured at a density of2×10⁵ per ml in Iscoves' Modified Dulbecco's Medium (Gibco, GrandIsland, N.Y.) in 0.3% (W/v) agar (FMC, Rockland, Me.), supplemented with25% fetal bovine serum (Hyclone, Logan, Utah), and 10⁻⁴2-mercaptoethanol in 35 mm culture dishes (Nunc, Naperville, Ill.).Cells were cultured in the presence of human IL-3, IL-6, G-CSF, GM-CSF,SCF at 100 ng/ml and EPO at 10 U/ml. Cultures were incubated at 37° C.in 5% CO₂ in a humidified incubator. At day 14 of culture, colonies wereenumerated using an inverted microscope. Macroscopic BFU-E were definedas those greater than 0.5 mm in diameter.

Marrow CFU-GM and BFU-E were assayed from four baboons before and at theend of the SCF infusion. The number of colonies per 10⁵ cells, i.e.,CFU-GM (41+/−12 pre-SCF, 36+/−post-SCF) and BFU-E (78+/−28 pre-SCF,52+/−26 post-SCF), were not statistically different. Given the dramaticincreases in marrow cellularity, the absolute numbers of CFU-GM andBFU-E were estimated to be increased.

A fifth baboon given SCF was studied weekly for changes in peripheralblood and marrow colony-forming cells. In marrow, the incidence ofCFU-GM increased 1.1 to 1.3 fold and BFU-E increased 2.5 to 6.5 fold. Inperipheral blood, however, the incidence of colony-forming cells wasmarkedly increased (25 to 100 fold), and absolute numbers ofcolony-forming cells were increased up to 96 fold for CFU-GM, 934 foldfor BFU-E, and greater than 1000 fold for the most primitivecolony-forming cells, CFU-MIX. This expansion of colony-forming cellswas apparent after as little as seven days of SCF administration and wasmaintained throughout the period that SCF was given.

B. Use of SCF in Bone Marrow Transplantation

As noted above, there are several ways that SCF is useful to improvehematopoietic transplantation. One method, as illustrated above is touse SCF to augment the harvest of bone marrow and/or peripheral bloodprogenitors and stem cells by pretreating the donor with SCF. Anotheruse is to treat the recipient of the transplanted cells with SCF afterthe patient has been infused. The recipient is treated with SCF alone orin combination with other early and late acting recombinanthematopoietic growth factors, including EPO, G-CSF, GM-CSF, M-CSF, IL-1,IL-3, IL-6, etc.

SCF alone enhances hematopoietic recovery following bone marrowtransplantation. A variety of experimental variables have been tested ina canine model of bone marrow transplantation, Schuening et al., 76636-640. in one set of experiments for the present invention, dogsreceived either G-CSF or SCF after 920 cGy of total body irradiation and4×10⁸ mononuclear marrow cells per kilogram from a DLA-identicallittermate. The hematologic recovery, as measured by day of neutrophilrecovery to 500 or 1000/mm³, is accelerated when either SCF or G-CSF isadministered compared to control animals that received no growth factor(Table 21). Recovery was 2-6 days earlier in animals that received SCFthan it was in those that received no growth factor. As noted above,combinations of appropriate growth factors with SCF will accelerate andenhance the response to those growth factors following hematopoietictransplantation.

TABLE 21 Effects of rcG-CSF and SCF on Recovery From DLA-indenticalLittermate Marrow Transplantation¹ Recovery of Recovery of TreatmentANC >500 mm³ ANC >1000/mm³ Control Day 10 Day 14 rcG-CSF² Day 7  Day 8 rcSCF³ #1 Day 7  Day 8  rcSCF³ #2 Day 8  Day 9  ¹920 cGY TBI followed byinfusion of 4 × 10⁸ mononuclear cells per kg DLA-identical lettermatebone marrow ²rcG-CSF administered 10 μg/kg/day_(SC) for 10 daysfollowing transplant ³rcSCF administered 200 μg/kg/day_(SC) for 10 daysfollowing transplant

This aspect of SCF in vivo biological activity can be utilized toenhance the recovery from marrow ablative therapy if the peripheralblood or bone marrow is harvested after SCF administration and thenre-infused after the ablative regimen (i.e., in bone marrowtransplantation or peripheral blood autologous transplantation).

EXAMPLE 23 Effect of SCF on Platelet Formation

Balb/c mice (female, 6-12 weeks of age, Charles River) were treated withrratSCF-PEG (100 ug/kg/day) or excipient control, subcutaneously, 1 timedaily for 7 days (n=7). Blood was sampled through a small incision inthe lateral tail vein on the indicated days after cessation of SCFtreatment. Twenty microliters blood were collected directly into 20 ulmicrocapillary tubes and immediately dispensed into the manufacturersdiluent for the Sysmex Cell Analyzer. Data points are the mean of thedata, error bars are standard error of the mean. Blood platelet countswere determined at the time points indicated in FIG. 53. Platelet countsrose to approximately 160% of control values by Day 4 post-SCF, fell tonormal by Day 10, and rose again to 160% of normal by Day 15. Plateletcounts stabilized at control values by Day 20.

A dose response curve of the SCF effect on platelet counts was generatedwhen Balb/c mice were treated as above with 10, 50, or 100 ug/kg/dayrratSCF-PEG (n=7). Blood was collected and analyzed on the fourth dayfollowing cessation of SCF treatment. These data are shown in FIG. 54and demonstrate that concentrations of rratSCF-PEG between 50-100ug/kg/day are optimal in inducing a rise in platelet counts. Recombinantrat SCF-PEG administration to normal mice also resulted in an increasein platelet size and in the number of megakaryocytes found in the spleenand bone marrow (Table 22). Rodent megakaryocytes were identified byexpression of the enzyme acetylcholinesterase (ACH+) which was detectedby cytochemical assays, [Long, Blood 58:1032 (1981)].

Certain similarities were noted between mice given SCF and mice duringrebound thrombocytosis after experimental induction of thrombocytopenia.FIG. 55 demonstrates one model of experimental thrombocytopenia, namelythat of treatment of 5-fluorouracil (5-FU). Balb/c mice were eitheruntreated or treated intravenously with 5-fluorouracil (150 mg/kg) onDay 0 (n=5). Blood analyses were performed on the indicated days as inlegend to FIG. 53. Error bars are present, but not discernable, in someof the control points. As has been demonstrated in the past [Radley etal., Blood 55:164 (1980)], animals become thrombocytopenic by Day 5post-5-FU. However, by Day 12 animals were in a state of reboundthrombocytosis where platelet counts far exceed normal (the “overshoot”effect). After Day 12, platelet counts appeared to cycle from normal tohigh levels throughout the 40 day testing period. As shown in FIG. 56,megakaryocyte numbers also rise dramatically after 5-FU appearing firstin the bone marrow (Panel A) and then in the spleen (Panel B). Themegakaryocyte numbers were determined in parallel with that shown inFIG. 55. Two Balb/c mice per group were sacrificed at the indicateddays. Cells from bone marrow (Panel A) or spleen (Panel B) werealiquoted at 100,000/well of a microtiter plate and stained foracetylcholinesterase according to published procedures, Long et al.,Immature megakaryocytes in the mouse: Morphology and quantitation byacetlycholinesterase staining. Blood 58: 1032, 1981. Data points are thepercentage of ACH+ cells per well for individual animals.

Platelet volumes also increase after 5-FU (FIG. 57). The data in thisfigure were generated from the same blood samples collected in FIG. 55.Mean Platelet Volume (MPV) is one of the parameters analyzed by theSysmex Cell Analyzer.

The possibility of a relationship between SCF and the physiologicalregulator of platelet production induced in the 5-FU thrombocytopenicmodel was explored. 5-FU was given to normal mice and SCF mRNAexpression levels quantitated in bone marrow cells collected on the daysindicated in FIG. 58. In FIG. 58, one million cells were lysed in SDSbuffer and the lysate was analyzed for the presence of mRNA specific formurine SCF. Probes for mouse SCF or human actin mRNA (which detects thecorresponding murine mRNA) were generated by runoff transcription ofcloned gene regions in vectors containing SP6 or T7 promoters using³⁵S-UTP according to standard protocols (Promega Biotech), or fromsynthetic oligonucleotide partial duplexes, Mulligan et al., Nuc. AcidsRes. 15:8783 (1987). RNA sense strand standards for quantitation of thehybridization assays were produced by runoff transcription of the sameregion in the direction opposite to the direction of probe synthesisusing tracer quantities of ³⁵S-UTP and 0.2 mM unlabeled UTP.

SCF or actin mRNA levels were quantitated as follows. Bone marrow cellswere explanted from animals at the given time post-5FU, enriched forlight density cells by centrifugation on 65% Percoll (Pharmacia;Pistcataway, N.J.) and lysed at 3×10⁶ nucleated cells/ml in 0.2% SDS, 10mM Tris pH 8, 1 mM EDTA, 20 mM dithiothreitol and 100 ug/ml proteinase K(Boerhinger Mannheim; Indianapolis, Ind.). Samples (30 ul) were added to70 ul of hybridization mix consisting of 30 ug/ml yeast tRNA, 30 ug/mlcarrier DNA, 145,000 CPM/ml ³⁵S-labeled probe in 3.0-3.7 M sodiumphosphate, pH 7.2 (depending on length of probe). Samples were incubatedat 84° C. for 2-3 hours then cooled to room temperature before additionof RNase A to 0.03 mg/ml and RNase T1 to 5000 U/ml. Samples wereincubated at 37° C. for 20 minutes before addition of 120 ul of 0.0025%bromophenol blue in formamide. Entire sample was then loaded onto 3.8 mlSephacryl S200 Superfine gel filtration column (0.7 cm×10 cm) and elutedwith 2.0 mls of 10 mM Tris pH 8, 1 mM EDTA, 50 mM NaCl. Effluentscontaining hybridized RNA duplexes were collected directly intoscintillation vials. After addition of 5 mls Liquiscint (New EnglandNuclear; Boston, Mass.) samples were counted 20 minutes or to 3% error.CPM were converted to molecules mRNA by comparison to the linear portionof the standard curve (correlation coefficient=0.97). The data point foreach sample is the mean of replicate tests; bone marrow samples from 3individual animals were taken for each time point so that the data shownis the mean of those determinations. Error bars are standard error ofthe mean. Statistical significance is assigned as described above.

SCF mRNA levels rose dramatically at Days 5 and 7, coinciding exactlywith the nadir of platelet counts immediately preceding thrombocytosis(FIG. 58).

The data in this section show that SCF is active as a thrombopoieticagent in vivo and furthermore that SCF may be involved in thephysiological regulation of platelet production after 5-FU-inducedthrombocytopenia.

TABLE 22 Megakaryocyte and platelet parameters measured on fourth dayfollowing SCF administration in vivo. % Ach + % Ach + Platelet Cells inCells in Factor Count MPV* Spleen Marrow none 1018 +/− 29 6.07 +/− 0.5.22 +/− .3 .02 +/− .01 SCF** 1429 +/− 56 6.24 +/− .05 .85 +/− .9 .59 +/−.05 *MPV; mean platelet volume **ratSCF-PEG administered SC 2 × dailyfor 7 days at 100 μg/kg/day. Data was collected 4 days later after lastinjection.

EXAMPLE 24 Treatment of Bone Marrow Failure States

A variety of congenital and acquired disorders of hematopoiesis havebeen reported to cause clinically significant reductions in the numberof mature circulating peripheral blood cells of one or more lineages.Therefore, the existing data supports that these disorders are treatablewith SCF. For example, aplastic anemia is a clinical syndromecharacterized by pancytopenia due to reduced or absent production ofblood cells in the bone marrow. It is heterogeneous in severity,etiology and pathogenesis. Most attention has focused on abnormalitiesof the hematopoietic stem cell, microenvironment or immunologic injuryof one of these. The response to immunosuppressive therapy is variableand incomplete. Because aplastic anemia is a defect of the hematopoieticstem cell or proliferative signals from the microenvironment, and ismodeled by the Steel mouse [Zsebo et al., Cell 63 213 (1990)], thisdisorder is successfully treated with SCF.

Another bone marrow failure disorder which is responsive to SCF isDiamond-Blackfan anemia (DBA) or congenital pure red cell aplasia. Thiscongenital abnormality results in a selective defect in the productionof red blood cells and often results in chronic transfusion dependency.In vitro data indicate that the defect is overcome by the addition ofexogenous SCF. Bone marrow from patients with DBA (or control marrow)was cultured with or without SCF (100 ng/ml) in the presence oferythropoietin (EPO) (1-5 U/ml), EPO plus IL-3 (1-1000 U/ml), EPO plusGM-CSF (>100 U/ml), or EPO plus lymphocyte-conditioned media (2-5%).Culture of bone marrow from patients with DBA demonstrate two patternsof response to SCF. The majority were hyper-responsive to SCF and showedapproximately 3 fold increase in the frequency of BFU-E at less than orequal to 10 ng/ml, as well as an increase in the size of BFU-E atconcentrations up to 200 ng/ml. Control marrow demonstrated only a 1.5fold increase in frequency of BFU-E. This pattern of response to SCFcould indicate a defect in endogenous SCF and/or its production by themicroenvironment in this group of patients with DBA. The other group ofpatients with DBA demonstrated an increase in the frequency of BFU-E atconcentrations of SCF greater than or equal to 50 ng/ml. This pattern ofresponse reflects an intrinsic defect in the receptor for SCF (c-kit) onthe progenitor cell. In either case (abnormal production of SCF by themicroenvironment or decreased stimulation of the hematopoieticprogenitor by SCF) SCF overcomes the block to hematopoiesis whichcharacterizes bone marrow failure syndromes such as DBA.

Other bone marrow failure syndromes that are treatable with SCF include,but are not limited to: Fanconi's anemia, dyskeratosis congenita,amegakaryocytic thrombocytopenia, thrombocytopenia with absent radii,and congenital agranulocytosis (e.g. Kostmann's syndrome,Shwachman-Diamond syndrome) as well as other causes of severeneutropenia such as idiopathic and cyclic neutropenia. Severe chronicneutropenia congenital, cyclic or idiopathic are treatable withrecombinant G-CSF.

Cyclic neutropenia, in particular, is a defect in the regulation of stemcell division since other lineages (e.g., platelet, erythrocyte andmonocyte) are also effected. In the canine model of cyclic neutropenia,the cycling of neutrophils, as well as other lineages, is sharplyreduced or even eliminated by SCF treatment. A typical dog with cyclicneutropenia was treated with rcanineSCF (recombinant canine SCF) at 100mg/kg/day subcutaneously over several weeks. The typical 21 day cyclefor neutrophils was eliminated during the first predicted cycle and thesecond predicted nadir was significantly atenuated. This is in contrastto treatment with G-CSF which increases the frequency and amplitude ofneutrophil cycling, but does not eliminate it. Thus, SCF is useful intreating a variety of bone marrow failure syndromes, either alone or incombination with other hematopoietic growth factors.

EXAMPLE 25 SCF Treatment of Patients with HIV-1 Infection

A. Source and Preparation of Peripheral Blood Mononuclear Cells

Leukopaks were obtained from HIV-, CMV-, and EBV-seronegative normaldonors from the American Red Cross. Peripheral blood was obtained from 6patients with HIV-infection after informed consent was obtained. Twopatients were asymptomatic, one had AIDS-related complex and three hadAIDS. None of the 6 patients had received zidovudine within the last sixmonths. None of the patients were anemic (hemoglobin <135 g/L) at thetime of study. All studies were conducted in accordance with UCLA HumanSubject Protection Committee regulations.

Peripheral blood mononuclear cells were isolated from leukopaks andperipheral blood using ficoll-hypaque sedimentation followed byextensive washing with Hank's Balance Salt Solution (HBSS). Blood cellswere enumerated and viability ascertained by trypan blue dye exclusion.

B. Burst Forming Unit Erythro (BFU-E) Assay

Assays for BFU-E were performed in a standard protocol using normalhuman bone marrow as the control. Heparinized blood was diluted with anequal volume of HBSS (GIBCO, Grand Island, N.Y.), layered overFicol-Paque (Pharmacia, Piscataway, N.J.) and centrifuged at 400 g for30 minutes at room temperature. Light density cells (s.g. <1.077) werecollected and washed twice in HBSS. Cells were resuspended in Iscove'sMedium with 10% Petal Bovine Serum (GIBCO, Grand Island, N.Y.) at aconcentration of 1×10⁷/ml. Cells (1×10⁵) were cultured in Iscove's Mediasupplemented with 5×10⁻⁵ M 2-Mercaptoethanol (2ME) (Sigma Chemicals, St.Louis, Mo.), 30% Fetal Bovine Serum (GIBCO, Grand Island, N.Y.), andeither 1 or 4 units of human recombinant erythropoietin (Amgen Inc.,Thousand Oaks, Calif.) in 0.3% agar. Four concentrations of E. coliderived human stem cell factor (hSCF¹⁻¹⁶⁴), obtained as described inExamples 6 and 10, were added (0, 10, 100 and 1000 ng/ml). Zidovudine(AZT) was added to the mixture resulting in final concentrations of 0,0.01 μM, 0.1 μM, 1.0 μM. Erythroid burst colonies were scored after 14days of culture in a humidified atmosphere containing 5% CO₂. Each assaywas done in duplicate and colonies with >50 cells present on day 14 withhemoglobinization were scored as BFU-E.

The 50% inhibitory concentration for zidovudine was calculated byexpressing the mean of four determinations of BFU-E for each level ofzidovudine and huSCF as a percentage of control (no added zidovudine).Linear regression was used to calculate the slope of inhibition. The 50%inhibitory concentration was calculated by interpolation and the valueused as the exponent for the base of 10. This results in directcalculation of the ID₅₀. The r² for all the slopes were >0.90.

C. Effects of HuSCF on Stimulated Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells were isolated from the leukopaks oftwo additional normal donors as described above. Cells were resuspendedin Iscove's Modified Dulbecco's Medium containing 20% fetal bovineserum, penn/strep, 1.0% PHA (Sigma Chemical, St. Louis, Mo.) and 10units/ml of interleukin-2 (Amgen Inc. Thousand Oaks, Calif.). Fourconcentrations of human stem cell factor (0, 10, 100, 1,000 ng/ml) wereadded to the media. Complete lymphocyte subset analysis of cellularantigens were analyzed in duplicate by two color fluorescent cytometryon day 0, 3, 7 and 10. Differences in percentages of cell populationswere detected using independent and paired t-tests (2-tailed).Comparisons were made between drug-treated and non-drug-treated valuesfor a single day and between single days values and baseline. Cytometricanalysis was done in duplicate.

D. Results

Exposure of peripheral blood mononuclear cells to erythropoietin andhuman stem cell factor (HuSCF) resulted in a dose-dependent increase inBFU-E formation in the 2 normal patients studied (FIG. 59A). Significantincreases (up to 100%) were seen with concentrations of human stem cellfactor between 10 and 1,000 ng/ml. Near maximal activity was seen at 10ng/ml suggesting that lower concentrations may be active. There weresignificant increases in BFU-E when the dose of erythropoietin wasincreased from 1 IU to 4 IU/ml (FIG. 59B). The colonies observed weresignificantly larger in size than the bursts seen in the absence ofHuSCF.

In the 6 HIV-infected individuals studied, significant dose-dependentincreases in BFU-E were also seen with HuSCF treatment (FIG. 60).Although the number of BFU-E in the absence of HuSCF was markedlyreduced compared to normal (range 2-26 BFU-E/10⁵ peripheral bloodmononuclear cells compared to approximately 74 BFU-E/10⁵ PBMC fornormals), the percentage increases in BFU-E were significantly higher inthe HIV-infected individuals. Near normal numbers of BFU-E were obtainedfor 2 individuals at the 1,000 ng/ml concentration of HuSCF. Althoughthe absolute number of BFU-E seen for some of the patients were stillwell below normal, all 6 individuals responded in vitro to HuSCF.

Because previous studies showed that cytokines could alter theintracellular uptake or intracellular metabolism of deoxynucleosides.[Perno et al., J. Exp. Med. 169:933(1989)] the capacity of hSCF tomodulate the inhibition of red cell progenitors by zidovudine wasevaluated. Each of the normals and all of the HIV individuals had BFU-Eassays performed in the presence and absence of 3 concentrations ofzidovudine and 4 concentrations of huSCF. As observed, (FIG. 59 and sizeof BFU-E bursts) the addition of HuSCF markedly reduced inhibition ofearly red cell progenitors by zidovudine. Significant alterations in the50% inhibitory dose of zidovudine for BFU-E was seen at all threeconcentrations of human stem cell factor. The IC₅₀ (fifty percentinhibitory concentration) ranged from 2.65 to 1376 μM of zidovudine(FIG. 61). All three of these inhibitory concentrations of zidovudineare well above normal serum levels obtained after 1,000 mg/day ofzidovudine [Klecher et al., Clin. Pharmacol. Ther.; 41:407-12 (1987)].Similar results were observed for all 6 individuals infected with HIV.However, because of the few number of red cell progenitors in 2 of thepatients, the increases in the 50% inhibitory concentrations ofzidovudine for BFU-E did not reach statistical significance.Nonetheless, the trends were clearly present and replicated the effectsof human stem cell factor on BFU-E in the presence of zidovudine in thenormal individuals.

The effect of SCF on the protection of bone marrow derived cells as wellas peripheral blood progenitors (above) was examined. Normal human bonemarrow was prepared as described above for peripheral blood progenitors.Bone marrow cells were exposed to different concentrations of AZT(zidouvidine), and the protective effects of SCF for both erythroid aswell as myeloid cells was determined in semi-solid cultures. Colonieswere scored after 14 day incubation as described above. The results forthe protection of bone marrow derived erythroid cells (FIG. 62) andmyeloid cells (FIG. 63) are indicated. As is seen for peripheral blood,SCF protects bone marrow cells from AZT as well. Another toxic compoundused to fight the opportunistic infections associated with HIV infectionis ganciclovir. Once again, SCF protects bone marrow cells against thetoxic effects of ganciclovir for both erythroid development (FIG. 64)and myeloid development (FIG. 65).

In summary, this example details the effects of HuSCF on early red bloodcell progenitors. Exposure to HuSCF in vitro resulted in a dose andtime-dependent increase in red blood cell progenitors and significantlyaltered the inhibition of red cell progenitors by zidovudine. This wasobserved in both normal and HIV-infected study populations. HuSCF had noeffect on HIV virus replication in primary monocytes or primary humanlymphocytes nor did it alter the efficacy of 2′,3′,-dideoxynucleosideanalogues. This is a significant difference from other cytokines whichhave effects on red cell progenitors such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). As shown inother studies [Koyanagi et al., Science 241:1773 (1981); Folks et al.,Science 238:800 (1987); Hammer et al., Proc. Natl. Acad. Sci. USA83:8734 (1986)], both GM-CSF and IL-3 significantly increase replicationof HIV in partially purified primary peripheral blood monocytes.

These studies demonstrate that human stem cell factor (HuSCF) is anideal candidate drug for use as adjunctive therapy in the treatment ofHIV-related pancytopenia. This cytokine appears to directly stimulatehuman hematopoietic progenitor cells and synergizes with IL-7, G-CSF,GM-CSF, and IL-3 in the production of pre-B lymphocytes, megakaryocytes,monocytes, granulocytes, and mast cells [Martin et al., Cell 63:203-211(1990); Zsebo et al., Cell, 63:213-224 (1990)].

EXAMPLE 26 Use of Stem Cell Factor to Facilitate Gene Transfer intoHematopoietic Stem Cells

The in vitro survival and proliferation of primitive stem cells iscritical to the success of gene transfer mediated by retroviralinsertion or other known methods of gene transfer. The effect of SCF onthe in vitro maintenance and/or proliferation of primitive progenitorcells has been studied in two systems which have been describedpreviously [Bodine et al., Proc. Natl. Acad. Sci. 86 8897-8901, 1989].The first is a pre-CFU-S assay wherein bone marrow cells are incubatedfor up to six days in suspension culture in the presence of growthfactors. Aliquots are injected into lethally iradiated mice and the micesacrificed at 12-14 days for quantitation of spleen focus formation.IL-3 and IL-6 synergize in enhancing the proliferation of CFU-S between2-6 days in culture. The second is a competitive repopulation assaywhich measures the effects of growth factors on recovery and biologicalactivity of cells capable of sustaining long-term hematopoiesis. Cellsfrom two congenic strains of mice differing for a hemoglobin marker areincubated in suspension independently, cells from one strain as acontrol and cells from a second under experimental conditions. Afterincubation, equal numbers of bone marrow cells from both cultures aremixed and injected into W/W^(v) recipients.

Rat SCF has been evaluated both in the pre-CFU-S and competitiverepopulation assays. SCF alone has very little activity in the pre-CFU-Sassay, similar to IL-3 alone. For enhancing CFU-S activity, thecombination of SCF and IL-3 is equivalent to the previous optimalcombination of IL-3 and IL-6 whereas the combination of SCF and IL-6 is5-fold more active than IL-3 and IL-6 (FIG. 66). A most advantageouscombination is SCF, IL-3 and IL-6; it is 6-fold more active than thecombination of IL-3 and IL-6.

In the competitive repopulation assay, the repopulating ability of cellscultured in the combination of SCF and IL-6 is superior at 35 days(short-term reconstitution) (FIG. 67). A most advantageous combinationfor long term reconstitution is SCF, IL-3 and IL-6, approximately1.5-fold greater than any combination of two factors. Based on thesedata, a most advantageous combination of soluble growth factors forenhancing retroviral mediated gene transfer into stem cells would beSCF, IL-3 and IL-6.

SCF presentation by stromal cells induces the proliferation of primitivebone marrow progenitors. The ultimate in vitro stimulus forproliferation of stem cells is provided by stromal cell linestransfected with human SCF cDNAs with sequences as shown in FIGS. 42 and44. When human bone marrow is cultured on artificial feeder layersexpressing the membrane bound form of human SCF 220 (FIG. 44), there isa continued proliferation of hematopoietic progenitors over time. Anexample of this is given in Table 23. Stromal cells derived from S1/S1embryos prior to their death in utero [Zsebo et al., Cell 63 213 (1990)]were transfected with human SCF cDNAs (either expressing the 220, FIG.44 or 248, FIG. 42, amino acid forms of SCF] and used as feeder layersfor human marrow. Briefly, adherent layers were treated with mitomycin Cand plated at confluence in 6 well plates. Normal human bone marrow,7.5×10⁵ adherence depleted cells, were plated in 5 ml of Iscove'sModified Dulbeccos Medium (Gibco), 10% fetal calf serum, and 10-6 Mhydrocortisone onto the transfected adherent layers. At the indicatedtime points, cells were withdrawn and plated in semi-solid agar usingEPO and IL-3 as a stimulus. For the experiment in Table 24, normaladherence depleted human bone marrow was first enriched forhematopoietic progenitors expressing the CD34 antigen using magneticparticle concentration [Dynal, Inc., Great Neck, N.Y.] prior to platingon the adherent feeder cells. In this case, 3.5×10⁴ cells were culturedon top of the adherent layers as described above. At the indicated timepoints, cells were withdrawn from the cultures and plated in semi-solidagar as described above. For both experiments, colony formation wasenumerated after 14 days of culture in a humidified atmosphere. Thegeneration of colony forming cells over time was enumerated. As isindicated, the membrane bound form of SCF (220 amino acid, FIG. 44) ismore potent at supporting hematopoiesis over time.

The S1/S1 cell line expressing human SCF¹⁻²²⁰ amino acid form isadvantageous for retroviral mediated gene transfer into hematopoieticstem cells. Human bone marrow is infected with retrovirus in thepresence of mammalian cells expressing human SCF¹⁻²²⁰. In addition, theviral producer line optimally is transfected with the human SCF¹⁻²²⁰gene and used for the viral infection as a co-culture.

TABLE 23 Generation of colony forming cells from normal human bonemarrow by cells expressing different splice variants of human SCF. Daysof Cells Culture CFU-Macs CFU-GM BFU-E CFU-Mix S1/S1-4  7 1.3 +/− 1    6+/− 3 3 +/− 3 0 14 0 0 0 0 21 0 0 0 S1/S1-4  7 31 +/− 13 51 +/− 8 3 +/−2 0 SCF 220 14 57 +/− 2  69 +/− 5 0 0 21 46 +/− 16  23 +/− 13 0 0S1/S1-4  7 57 +/− 14 89 +/− 7 11 +/− 8  1 +/− 1 SCF 248 14 5 +/− 4  9+/− 5 5 +/− 3 0 21 1 +/− 1 0 0 0

TABLE 24 Generation of colony forming cells from CD34+ bone marrow cellsexpressing different splice variants of human SCF. Days of Totalcolonies/culture well Cells Culture CFU-Macs CFU-GM BFU-E CFU-MixS1/S1-4  7  4 +/− 2 10 +/− 6 11 +/− 3   +/− 1 14 0 0 0 0 21 0 0 0 0S1/S1-4  7 90 +/− 7 70 +/− 2 18 +/− 10 13 +/− 4 SCF 220 14  14 +/− 13 60 +/− 11 2 +/− 1 0 21 36 +/− 3 23 +/− 5 0 0 S1/S1-4  7 260 +/− 64 135+/− 20 80 +/− 20 15 +/− 5 SCF 248 14 0 0 0 0 0 0 0

EXAMPLE 27 Further Characterization of Recombinant Human SCF Obtainedfrom E. coli or CHO Cells

As noted in Example 10, human [Met⁻¹]SCF¹⁻¹⁶⁴ from E. coli has aminoacid composition and amino sequence expected from analysis of the gene.Using the methods outlined in Example 2, it has been determined thathuman SCF¹⁻¹⁶⁵ obtained from E. coli as described in Example 10 also hasthe amino acid composition and amino acid sequence expected fromanalysis of the gene, and also retains Met at position (−1).

Purified E. coli-derived human [Met⁻¹]SCF¹⁻¹⁶⁴ and CHO cell-derivedhuman [Met⁻¹]SCF¹⁻¹⁶² have been studied by methods indicative ofsecondary and tertiary structure. Fluorescence emission spectra, withexcitation at 280 nm, have been obtained. These are shown in FIG. 68.The molecules were dissolved in phosphate-buffered saline. The spectraconsist of a single peak with a maximum at 325 nm, and a full width athalf maximum (FWHM) of between 45 and 50 nm. Both the emissionwavelength and the FWHM suggest that the single Trp is present in ahydrophobic environment, and that this environment is the same in bothmolecules.

Circular dichroism studies have also been carried out. FIG. 69 shows thefar ultraviolet (UV) spectra and near UV spectra (B) for the E.coli-derived SCF (solid lines) and CHO cell-derived SCF (dotted lines).The molecules were dissolved in phosphate-buffered saline. The far UVspectra contain minima at 208 nm and 222 nm. Using the Greenfield-Fasmanequation [Greenfield and Fasman, Biochemistry 8, 4108-4116 (1969)], thespectra suggest 47% α-helix, while the method of Chang et al. [Anal.Biochem. 91, 13-31 (1978)] indicates about 38% α-helix, 33% β-sheet, and29% disordered structure. The near UV spectra have minima at 295 nm and286 nm attributable to tryptophan, minima at 261 nm and 268 nmattributable to phenylalanine, and minima at 278 probably attributableto tyrosine, with some overlap between chromophores. The resultsindicate that the aromatic chromophores are located in asymmetricenvironments. Both the far UV and near UV results are the same for E.coli-derived SCF and CHO cell-derived SCF, indicating similarity ofstructure.

Second derivative infrared spectra in the amide I region (1700-1620cm⁻¹) of the E. coli-derived SCF (A) and CHO cell-derived SCF (B) areshown in FIG. 70. These spectra are related to polypeptide backboneconformation [Byler and Susi, Biopolymers 25, 469-487 (1986); Surewiczand Mantsch, Biochim. Biophys. Acta 952, 115-130 (1988)] and areessentially identical for the two proteins. Band assignments [Byler andSusi (1986), supra; Surewicz and Mantsch (1988), supra] allow one toestimate that the two SCFs have predominantly helical structures, −31%α-helix and 19% 3₁₀-helix, with lesser fractions of β-strands (˜25%),turns (˜15%), and disordered structures (˜14%).

Disulfide structure of various molecules referred to in previousexamples have been determined. These include BRL 3A cell-derived naturalrat SCF, E. coli-derived rat [Met⁻¹]SCF¹⁻¹⁶⁴, CHO cell-derived ratSCF¹⁻¹⁶² , E. coli-derived human [Met⁻¹]SCF¹⁻¹⁶⁴ , E. coli-derived human[Met⁻¹]SCF¹⁻¹⁶⁵, and CHO cell-derived human SCF¹⁻¹⁶². The methods usedinclude those outlined in Example 2 for amino acid sequence andstructure determination. The proteins are digested with proteases, andthe resulting peptides isolated by reverse-phase HPLC. If this is donewith and without prior reduction, it is possible to isolate and identifydisulfide-linked peptides. Isolated disulfide-linked peptides can alsobe identified by plasma desorption mass spectroscopy. By such methods ithas been demonstrated that all of the above-mentioned molecules haveintrachain disulfide bonds linking Cys-4 and Cys-89, and linking Cys-43and Cys-138.

EXAMPLE 28 Production and Characteristics of SCF Analogs and FragmentsExpressed in E. coli

Plasmid constructions for expression of numerous SCF analogs andfragments have been made. Site-directed mutagenesis has been used toprepare plasmids with initiating methionine codon followed by codons foramino acids 1 to 178, 173, 168, 166, 163, 162, 161, 160, 159, 158, 157,156, 148, 145, 141, and 137, using the numbering of FIG. 15C. The DNAfor human SCF¹⁻¹⁸³ (Example 6B) was cloned into MP11 from Xba1 to BamH1.Phage from this cloning was used to transfect an E. coli dut⁻ ung⁻strain, R21032. Single stranded M13 DNA was prepared from this strainand site-directed mutagenesis was performed (reference IL-2 patent).After the site-directed mutagenesis reactions, the DNAs were transformedinto an E. coli dut⁺ ung⁺ strain, JM101. Clones were screened andsequenced as described in copending U.S. patent application Ser. No.717,334, filed Mar. 29, 1985. Plasmid DNA preps were made from positiveclones and the SCF regions from Xba1 to BamH1 were cloned into pCFM1656as described in copending U.S. patent application Ser. No. 501,904,filed Mar. 29, 1990. The oligonucleotides for each cloning were designedto substitute a stop codon for an amino acid codon at the appropriateposition for each analog.

Plasmids with initiating methionine codon followed by codons for aminoacids 1 to 130, 120, 110, 100, 133, 127, and 123 (using the numbering ofFIG. 42) have been made using the polymerase chain reaction. ThepCFM1156 human SCF¹⁻¹⁶⁴ plasmid DNA (Example 6B) was used to prime thereaction using a 5′ oligonucleotide 5′ to the Xba1 site and a 3′oligonucleotide which included a direct match to the desired 3′ end ofthe analog DNA, followed by a stop codon, followed by a BamH1 site.After the polymerase chain reaction, the polymerase chain reactionfragments were cleaved with Xba1 and BamH1, gel purified, and clonedinto pCFM1656 cut with Xba1 and BamH1.

Plasmids with initiating methionine codon followed by codons for aminoacids 2 to 164, 5 to 164, and 11 to 164 (using the numbering of FIG. 42)were also made using polymerase chain reaction. The pCFM1156 humanSCF¹⁻¹⁶⁴ plasmid DNA (Example 6B) was used with two primers. The 5′oligonucleotide primer included an Nde1 site (which includes the ATGcodon for the initiating methionine) and a homologous stretch of DNAstarting at the codon for the first desired amino acids. The 3′oligonucleotide primer was totally homologous and was 3′ to the EcoR1site in the gene. After the polymerase chain reaction, the fragment wascut with Nde1 and EcoR1, gel purified, and cloned back into the pCFM1156human SCF¹⁻¹⁶⁴ plasmid cut with Nde1 and EcoR1.

A plasmid with initiating methionine codon followed by codons for aminoacids 1 to 248 (using the numbering of FIG. 42) was made using DNAobtained directly from the cDNA clone (Example 16). The cDNA was cleavedwith Spe1 and Dra1 (blunt end) and the fragment with the SCF region wasgel purified. This was cloned into the pCFM1156 human SCF¹⁻¹⁸³ plasmid(Example 6B) which had been cut with HindIII, end filled with the Klenowfragment of DNA polymerase 1 (to yield a blunt end), and then cut withSpe1 and gel purified. To allow for site-directed mutagenesis as above,the SCF¹⁻²⁴⁸ fragment was cloned into MP11 from Xba1 to BamH1; analogplasmids encoding initiating methionine followed by amino acids 1-189,1-188, 1-185, or 1-180 (using numbering of FIG. 42) were then made usingsite-directed mutagenesis.

A plasmid with initiating methionine codon followed by codons for aminoacids 1 to 220 (using the numbering of FIG. 44) was made using DNAdirectly from the cDNA clone (Example 18), using the same methodsoutlined in the preceding paragraph. Similarly, analog plasmids encodinginitiating methionine followed by amino acids 1-161, 1-160, 1-157, or1-152 (using the numbering of FIG. 44) were made.

A pCFM1156 human SCF²⁻¹⁶⁵ plasmid was made by cloning the Xba1 to EcoR1SCF fragment from pCFM1156 human SCF²⁻¹⁶⁴ into the plasmid pCFM1156human SCF¹⁻¹⁶⁵ (having synthetic codons; see Example 6B). Both DNAs werecut with Xba1 and EcoR1 and the fragments gel purified for cloning. Thesmall fragment from pCFM1156 human SCF²⁻¹⁶⁴ was ligated to the largefragment of pCFM1156 human SCF¹⁻¹⁶⁵ (synthetic codons).

In considering the analog plasmids described above, it is noted thatamino acids 4, 43, 89, and 138 are Cys in human SCFs, and the codons forCys-4 or Cys-138 are missing in certain of the plasmids described. Aminoacids of the hydrophobic transmembrane region are at positions 190(about) to 212 in the numbering of FIG. 42, and positions 162 (about) to184 in the numbering of FIG. 44. Thus most of the plasmids describedencode amino acids that would be in the extracellular domain of membranebound human SCF¹⁻²⁴⁸ (FIG. 42 numbering) or human SCF¹⁻²²⁰ (FIG. 44numbering), and some include virtually all of these extracellulardomains.

Plasmids encoding various other human SCF analogs and fragments can alsobe prepared by the methods described, and by other methods known tothose skilled in the art. These include plasmids with codons for Cysresidues replaced by codons for other amino acids such as Ser.

E. coli host strain FM5 (Example 6) has been transformed with many ofthe analog plasmids described. These strains have been grown, withtemperature induction, in flasks, and in fermentors as described inExample 6C.

After fermentation and harvesting of cells, many folded, oxidized,purified SCF analogs have been recovered by the methods outlined inExample 10. These include (by the numbering of FIG. 42) SCF¹⁻¹⁸⁹,SCF¹⁻¹⁸⁸, SCF¹⁻¹⁸⁵, SCF¹⁻¹⁸⁰, SCF¹⁻¹⁵⁶, SCF¹⁻¹⁴¹, SCF¹⁻¹³⁷, SCF¹⁻¹³⁰,SCF²⁻¹⁶⁴, SCF⁵⁻¹⁶⁴, SCF¹¹⁻¹⁶⁴, and (by the numbering of FIG. 44)SCF¹⁻¹⁶¹, SCF¹⁻¹⁶⁰, SCF¹⁻¹⁵⁷, SCF¹⁻¹⁵². Like SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵(Examples 17 and 27), these analogs are all dimeric in solution, asjudged using gel filtration. Most of these have biological specificactivities in the radioreceptor assay (Example 9) and UT-7 proliferationassay (Example 9) similar to those of SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵ (Example 9).Some, such as SCF²⁻¹⁶⁴ and SCF⁵⁻¹⁶⁴ have lowered specific activities inthe radioreceptor assay and/or UT-7 assay (30-80% of the values forSCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵) while others, such as SCF¹¹⁻¹⁶⁴, have negligiblespecific activity in both assays. SCF¹⁻¹³⁰ has lowered specific activityin both the radioreceptor assay (about 50% of the value for SCF¹⁻¹⁶⁴)and the UT-7 assay (about 15% of the value for SCF¹⁻¹⁶⁴). SCF¹⁻¹³⁷ hasfull specific activity in the radioreceptor assay but lowered specificactivity in the UT-7 assay (about 25% of the value for SCF¹⁻¹⁶⁴ andSCF¹⁻¹⁶⁵); this analog therefore may be preferable as an SCF antagonistin situations where it would be advantageous to block the biologicalactivity of SCF.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

104 165 amino acids amino acid single linear protein unknown 1 Glu GluIle Cys Arg Asn Pro Val Thr Asp Asn Val Lys Asp Ile Thr 1 5 10 15 LysLeu Val Ala Asn Leu Pro Asn Asp Tyr Met Ile Thr Leu Asn Tyr 20 25 30 ValAla Gly Met Asp Val Leu Pro Ser His Cys Trp Leu Arg Asp Met 35 40 45 ValThr His Leu Ser Val Ser Leu Thr Thr Leu Leu Asp Lys Phe Ser 50 55 60 AsnIle Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Gly 65 70 75 80Lys Ile Val Asp Asp Leu Val Ala Cys Met Glu Glu Asn Ala Pro Lys 85 90 95Asn Val Lys Glu Ser Leu Lys Lys Pro Glu Thr Arg Asn Phe Thr Pro 100 105110 Glu Glu Phe Phe Ser Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp 115120 125 Phe Met Val Ala Ser Asp Thr Ser Asp Cys Val Leu Ser Ser Thr Leu130 135 140 Gly Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe MetLeu 145 150 155 160 Pro Pro Val Ala Ala 165 26 base pairs nucleic acidsingle linear DNA unknown modified_base /mod_base= Inosine 2 ACRTTYTTNGGNGCRTTYTC YTCCAT 26 23 base pairs nucleic acid single linear DNAunknown modified_base 12 & 15 /mod_base= Inosine 3 AARAAYTCYT CNGGNGTRAARTT 23 14 base pairs nucleic acid single linear DNA unknown 4 GTYTCNGGYTTYTT 14 26 base pairs nucleic acid single linear DNA unknown 5ATGGARGARA AYGCCCCCAA RAAYGT 26 20 base pairs nucleic acid single linearDNA unknown 6 CCNAAYGAYT AYATGWTMAC 20 20 base pairs nucleic acid singlelinear DNA unknown 7 GGNGGNARCA TRAANGGYTT 20 23 base pairs nucleic acidsingle linear DNA unknown 8 ACCAKAARAT CTTYAAANCG ATC 23 22 base pairsnucleic acid single linear DNA unknown 9 GTATTTTCAA TAGATCCATT GA 22 14base pairs nucleic acid single linear DNA unknown 10 CCAACTATGT CGCC 1421 base pairs nucleic acid single linear DNA unknown 11 GTAGTCAAGCTGACTGATAA G 21 21 base pairs nucleic acid single linear DNA unknown 12TAACCAACAA TGACTAGGCA A 21 16 base pairs nucleic acid single linear DNAunknown 13 TTCCAGAGTC AGTGTC 16 29 base pairs nucleic acid single linearDNA unknown 14 GCGAAGCTTG CCTTTCCTTA TGAAGAAGA 29 38 base pairs nucleicacid single linear DNA unknown 15 GCGCCGCGGT TACGGTGGTA ACATGAAGGGCTTTGTGA 38 21 base pairs nucleic acid single linear DNA unknown 16GATAAATGCA AGTGATAATC C 21 36 base pairs nucleic acid single linear DNAunknown 17 GCGGTCGACC CGCGGAACTT TAAGTCCATG CAACAC 36 36 base pairsnucleic acid single linear DNA unknown 18 CACCCGCGGT TATGCAACAGGGGGTAACAT AAATGG 36 36 base pairs nucleic acid single linear DNAunknown 19 CACCCGCGGT TAGGCTGCAA CAGGGGGTAA CATAAA 36 18 base pairsnucleic acid single linear DNA unknown 20 CTTAATGTTG AAGAAACC 18 22 basepairs nucleic acid single linear DNA unknown 21 GATGGTAGTA CAATTGTCAG AC22 22 base pairs nucleic acid single linear DNA unknown 22 GTCTGACAATTGTACTACCA TC 22 22 base pairs nucleic acid single linear DNA unknown 23CAATTTAGTG ACGTCTTTTA CA 22 24 base pairs nucleic acid single linear DNAunknown 24 TTAGATGAGT TTTCTTTCAC GCAC 24 24 base pairs nucleic acidsingle linear DNA unknown 25 AAATCATTCA AGAGCCCAGA ACCC 24 18 base pairsnucleic acid single linear DNA unknown 26 AACATCCATC CCGGGGAC 18 29 basepairs nucleic acid single linear DNA unknown 27 CTGGCAATAT TTTAAGTCTCAAGAAGACC 29 29 base pairs nucleic acid single linear DNA unknown 28GCGCCGCGGC TCCTATAGGT GCTAATTGG 29 27 base pairs nucleic acid singlelinear DNA unknown 29 CCTCACCACT GTTTGTGCTG GATCGCA 27 31 base pairsnucleic acid single linear DNA unknown 30 GGTGTCTAGA CTTGTGTCTTCTTCATAAGG A 31 10 base pairs nucleic acid single linear DNA unknown 31CCCCCCCHGG 10 20 base pairs nucleic acid single linear DNA unknown 32TTTTTTTTTT TTTTTTTTGG 20 20 base pairs nucleic acid single linear DNAunknown 33 TTTTTTTTTT TTTTTTTTAG 20 20 base pairs nucleic acid singlelinear DNA unknown 34 TTTTTTTTTT TTTTTTTTCG 20 24 base pairs nucleicacid single linear DNA unknown 35 TTCGGCCGAT CAGGCCCCCC CCCC 24 30 basepairs nucleic acid single linear DNA unknown 36 TTCGGCCGGA TAGGCCTTTTTTTTTTTTTT 30 24 base pairs nucleic acid single linear DNA unknown 37GGCCGGATAG GCCTCACNNN NNNT 24 17 base pairs nucleic acid single linearDNA unknown 38 GGCCGGATAG GCCTCAC 17 4673 base pairs nucleic acid singlelinear DNA unknown CDS join(660..773, 1184..1246, 2053..2223,2837..2993, 3692..3774) mat_peptide join(720..773, 1184..1246,2053..2223, 2837..2993, 3692..3774) 39 AAAGTATCTT TCTATTGGCG AAGGACATGTTTTCCCATAA GTGGTAAACA AACTGTCTGC 60 ACATAATAAT TATCTTGCTG CCGTAAAGATTAGGTTAAAT TCTGCCTTCG ATCTAAAAAC 120 ACACCCTTCT GTCAATCCGA GGAGCAGTGTGCTAGTCTAG AGGTCTAAAT GAAGGCTCCT 180 TTCACGGTTG TATTTCTGCT CCCCAAATTGTCCACATTTA AAAGGAGAGT GCTTCTTTTC 240 AGCCTTAGGC TCTGAATTTC ATGCATTCCTCCATTTTCCG AGGTCCCCCC CAAGTGATAA 300 TTCTGTTACA CGTTGCTACA AGTTCATCCCTAATTGCCGT CAAGAAACTG ACTGTAGAAG 360 GCTTACCACA GACGTTGTAA CCGACAGTAAAGCCATTGAA AGAGTAATTC AAACAGGATG 420 GAAGCCAGGA GTATTTTGTG GCTGTTGCTCTTTTTCTTTT CAGTTTGGTG AGAGCAGCTT 480 GAATGCTTAA CATTTAAGCC ATCAGCTTAAAACAAAACAA AACAAAACAA AAAAAAACCC 540 CGCTCTGGCA TATTTGCACT TAACACATACGGTATAAGGT GTTACTGGTT TGCATAGTTC 600 TGGATTTTTT TTTTTTAAAA ACTGATGGACACCAAGAAAT GTTTCTGTTC TTTGTTTAG 659 ACT TGG ATT ATC ACT TGC ATT TAT CTTCAA CTG CTC CTA TTT AAT CCT 707 Thr Trp Ile Ile Thr Cys Ile Tyr Leu GlnLeu Leu Leu Phe Asn Pro -20 -15 -10 -5 CTC GTC AAA ACT CAG GAG ATC TGCAGG AAT CCT GTG ACT GAT AAT GTA 755 Leu Val Lys Thr Gln Glu Ile Cys ArgAsn Pro Val Thr Asp Asn Val 1 5 10 AAA GAC ATT ACA AAA CTG GTAAGTAAAGAATGATTTTG GCATCTATAA 803 Lys Asp Ile Thr Lys Leu 15 GTCTTCCCTGTGCTTGCTGA CCACATAGGT TCAGGGCACT CCCGACAGGA GTTCCCAGCT 863 TTCTAAGATAAGGAATCACT GTACGAGTCT GAAGTGCTTC TTCTGGGCAA ATGGGAGATG 923 CTTAGGTCATGGAGGGTTTA TCTGTATAAC TGGCCCTTTG CACACCAACA AAGTGACTGA 983 CTGGCTTTTGCCTGTTACCT ACTGTCTCCA GTCCTGGGCA TGGTATATAC TTAGGCACCC 1043 AAGATTGGATTTACAACTCA AGCATTATAT ATTGGACAAC ACGGGGTATG AGATATTAAT 1103 GATATGTCAGGTTGGATGGA TGAGTTTTCT CAAGAAATTC TCTTGTATTT ACTCACGTTT 1163 TCATTTCTTGGTCTCTGTAG GTG GCG AAT CTT CCA AAT GAC TAT ATG ATA 1213 Val Ala Asn LeuPro Asn Asp Tyr Met Ile 20 25 ACC CTC AAC TAT GTC GCC GGG ATG GAT GTTTTG GTATGTAGTC CACACACTTC 1266 Thr Leu Asn Tyr Val Ala Gly Met Asp ValLeu 30 35 TGAGTTGCCT TTTAGTAGCT AATGGGTGAC CTGTGCTTAT TCACATTGAAGACATTATTT 1326 GCTCTTTGTC GTTTTTAGAT GTTGACCTAT AATTTTTCCT TCAAGCTGCTGCTAAGATTA 1386 TCAGTGAGCA TTTCAGTATG TGTTTTAAGC CTACTCATTA AAAGGAAATGGCTCATCTTA 1446 GACGTAGCAA CCGATGTTAA TTTTTCCCCA GGCATCTCTC AGAGGGACTTGAATGTTAAA 1506 ATCATGTTAA ATTTCCTCCT TGGCTATGTT ATTTCTCATG GCTATGTTATTCCTATTCGT 1566 ATTTCATTTA AAGGGACGGA ATATTTATTG TATTTCTGAA CTTTTTCAGGCATGCATCCG 1626 GGTCTTTGAA TAAAACACTA AGACTCCTTC TAGTAATGTT TGTAATCCTGTCTGTATCGA 1686 ATGTCTTTGA AAACGCAGTG ACTAAGCCAT AAATAATCTT CCACAGAACGTCCAGTGGTT 1746 CATGAACTTT GTATGTGGGG GTGGGGCAAG AATTGTCTCA CTATTGGTCAAGGAAGAGAA 1806 GGTAAGGTAT GCAAGGGTGG TTTAATCTTC TTCCGTGGAA GGACAAAATCATCTATCATT 1866 TCCTCTGATC TCTATGCATT TGTTTGTTTT GAACTGAATC TGACTTGAGCAAGAGTTGGC 1926 GTCCTGTGTT CTGAGGAAAC TCTTTGTCCT GCAGTCAGTG ACTAAAAGTGCTGAGAGATC 1986 TGAAGAGCAC TCTGAATCTG CCATATTTTT AATAGATGCT TTGTCTTCTCTTTGAATTTC 2046 TTCCAG CCT AGT CAT TGT TGG TTA CGA GAT ATG GTA ACA CACTTA TCA 2094 Pro Ser His Cys Trp Leu Arg Asp Met Val Thr His Leu Ser 4045 50 GTC AGC TTG ACT ACT CTT CTG GAC AAG TTT TCA AAT ATT TCT GAA GGC2142 Val Ser Leu Thr Thr Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly 5560 65 TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GGG AAA ATA GTG GAT GAC2190 Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Gly Lys Ile Val Asp Asp 7075 80 85 CTC GTG GCA TGT ATG GAA GAA AAT GCA CCT AAG GTAACTTGGTATTCATCAGA 2243 Leu Val Ala Cys Met Glu Glu Asn Ala Pro Lys 90 95ATTATTTTTC TTATACTGAG CTCATGATGA GCAATTCACA ACCACTTGTA ATTCCAGCTC 2303CAGAGGACAT TATCCCCTCT TTGGATGCCA TAGGAATCTG CTCTCAAATA TGTAGATACC 2363ACCTCTGCCA CCTCAGCACA TACATACACA TAATTAAAAA ATAGAAACAT TAAAGGAGTT 2423CCAATCAATC CTTATTCTTT TCTGTATTCA GTATGCCCAG ATGTAAATTC TAGGAATATG 2483TTTTAAAGGC TAATTCTTAT TTTGTAATAA GCAGCTTTAA AATTCTTAAT TGTTTTTTCG 2543GGTCACTTTA TTGTCCTATT GCCACGACAT TGTCCTGTCC CATTGTCTGT TATTCCTTCT 2603GTTTTGTTTA TTGTTCCCTA GTTACTTTGA TCATGAGATT GACCTGTTAC CCGTTGTTAT 2663TCTCTGTAGC CATTTTGAGT TGTGTCTATT AGAACAGCTG TTAAATTACT TGAATCATTG 2723AGGACATAGT CAATAATCTA TTATGCTGAT CCAGTCAAGT CTATGAGTTA TTTGAAAACT 2783AGAATCTTTG TTAATTATTT GTTTGCTTGT TTGTTTGTTT ATTATTTGTC TAG AAT 2839 AsnGTA AAA GAA TCA CTG AAG AAG CCA GAA ACT AGA AAC TTT ACT CCT GAA 2887 ValLys Glu Ser Leu Lys Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu 100 105 110GAA TTC TTT AGT ATT TTC AAT AGA TCC ATT GAT GCC TTC AAG GAC TTC 2935 GluPhe Phe Ser Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe 115 120 125ATG GTG GCA TCT GAC ACT AGT GAT TGT GTG CTC TCT TCA ACA TTA GGT 2983 MetVal Ala Ser Asp Thr Ser Asp Cys Val Leu Ser Ser Thr Leu Gly 130 135 140145 CCT GAG AAA G GTAAGGCTTT TAAGCATTTC TTGTTTAAAT GTACATAGAA 3033 ProGlu Lys AGCCTGAACT TCTGTAAGCC TCTACTGCTG AATCAACTAA ATGTGTTGCTGTAGAAAGAA 3093 CGTGTGGGTT TTTCTGATAA AAACAAAAAG CAAATATCAA TGACTACCAATGATTATTAT 3153 CTAGCTTGAG AGATATGCCC TAAGACAGCG ATTCTCGATA TTTCTAAATTAAAGAATTGT 3213 GTGATGGTGG CTCACATATT TTCTAACTGT GATATTTGCC AGGAGAGTAGAATAATGTTA 3273 TTCTTCATCC CCAGAATTCC TAAGATTTCA CGTCTCATGT CTTTTCCATAAGGTTCAAAC 3333 TCTGAGACTT GAGTTCTGAG CCTCAGCAGG TCATTCTGAA TCCCCACTCTCCCCGAGCTG 3393 GGTCCCTATG GGGGAACTAA CTTCATTGCT TTCTTTTAAA ACATGACGAGTTACCAACAG 3453 CTCCTCGCTA TTATAAACAT GTTCCTAAGC ATGTCTGTGC ATGCAATAAGCCTTCACTCT 3513 ACAAGACAGT TATGGTGTAT CGCTTGACAA AACTGAGCAG CCAAGCTGAGTATGAAATAA 3573 TAATCTAGAC TTGGGAGGCA GACCCAGCAC CTACTGTGAT ATTGCACTTCGCCTTTGGGG 3633 GACTCTATGA TTCAAAAGTT CACCATGTAA CACTGACACA TTATTGCTTTCTATTTAG AT 3693 Asp TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA CCC CCTGTT GCA GCC 3741 Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro ValAla Ala 150 155 160 165 AGT TCC CTT AGG AAT GAC AGC AGT AGC AGT AATAGTAAGTACA CATATCTGAT 3794 Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn170 175 TTACTGCATG CATGGCTCCA AGTATCCTCT ATAGGAGTGT TGCATGGACTTAAAGTTTAT 3854 AAATCACTAC TAATAATGCT GTTCTGTCAC TGTTATTCCT TGTATGGGCTTCCTGACAAT 3914 TAAATATCTG GTTTGTAGAA TCGGATCTCC TTAGAGGTTA AGATGACCATGACAAAATTA 3974 GGCCAATCAA CTTTCTGCGA AGGTTATTTT AAATAAGGCA CGAAATTAATTGAAGGAAAA 4034 AAAAATACAA GCAAGGCCTT ATTTTGAATC ATGGTAGGCT TAAAATAGACTTTGTGGAGA 4094 ATGTCCCTGA TCAAAGTGGA GTTTTCAGAT TTCAAGTGCA TGTGCTAACTCTCCACAATG 4154 TCAAGGCTAT TTTCAGTTTT GTGTCTCCAT ATTTACTACT GCATGTTTGGAAATTTGCTG 4214 ATGCTGTTAG ATTACCTAAG AATGTATGTT GAAGAAGAAT GGACTTCTTTCCCTAAAATT 4274 TCTGTCCTCT TTGCCCAAGA ACCCACGTTC CTGGAAGACT ATCTTATTTTCATGTCTGTG 4334 CAATGATCAT TATAAAGATT ATTGAATATA CTGGGAATAC TCTGGTTTCTGTTTTTACAG 4394 ATTCATAATA GCTTATTCAG TCTTTAAAGA AAGTTCTCTG AAGTCCATGCTTTAGAATGT 4454 TTCTCTATCA AAACTTGACC TGGACCTTAA ATAAAGCTAT ATTTAGTCTTTTTATCCCTG 4514 AAAAATATAT TTCACAGTGT AGACATTTGA TATACATCTA AGGGAAGGATGCTGCCAGAA 4574 TGCTCGGGCT GGCAGTCTAC AAAGTCCACT GCTCTCAGGA TGGACTTCTGAAAGCGGAAA 4634 TTGTGAACTG CATGCATATA ACATATCAGA TCCTCGAGC 4673 196amino acids amino acid linear protein unknown 40 Thr Trp Ile Ile Thr CysIle Tyr Leu Gln Leu Leu Leu Phe Asn Pro -20 -15 -10 -5 Leu Val Lys ThrGln Glu Ile Cys Arg Asn Pro Val Thr Asp Asn Val 1 5 10 Lys Asp Ile ThrLys Leu Val Ala Asn Leu Pro Asn Asp Tyr Met Ile 15 20 25 Thr Leu Asn TyrVal Ala Gly Met Asp Val Leu Pro Ser His Cys Trp 30 35 40 Leu Arg Asp MetVal Thr His Leu Ser Val Ser Leu Thr Thr Leu Leu 45 50 55 60 Asp Lys PheSer Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile 65 70 75 Asp Lys LeuGly Lys Ile Val Asp Asp Leu Val Ala Cys Met Glu Glu 80 85 90 Asn Ala ProLys Asn Val Lys Glu Ser Leu Lys Lys Pro Glu Thr Arg 95 100 105 Asn PheThr Pro Glu Glu Phe Phe Ser Ile Phe Asn Arg Ser Ile Asp 110 115 120 AlaPhe Lys Asp Phe Met Val Ala Ser Asp Thr Ser Asp Cys Val Leu 125 130 135140 Ser Ser Thr Leu Gly Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys 145150 155 Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser160 165 170 Ser Ser Ser Asn 175 849 base pairs nucleic acid singlelinear protein unknown CDS 26..844 mat_peptide 101..844 41 CTGGATCGCAGCGCTGCCTT TCCTT ATG AAG AAG ACA CAA ACT TGG ATT ATC 52 Met Lys Lys ThrGln Thr Trp Ile Ile -25 -20 ACT TGC ATT TAT CTT CAA CTG CTC CTA TTT AATCCT CTC GTC AAA ACT 100 Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn ProLeu Val Lys Thr -15 -10 -5 CAG GAG ATC TGC AGG AAT CCT GTG ACT GAT AATGTA AAA GAC ATT ACA 148 Gln Glu Ile Cys Arg Asn Pro Val Thr Asp Asn ValLys Asp Ile Thr 1 5 10 15 AAA CTG GTG GCG AAT CTT CCA AAT GAC TAT ATGATA ACC CTC AAC TAT 196 Lys Leu Val Ala Asn Leu Pro Asn Asp Tyr Met IleThr Leu Asn Tyr 20 25 30 GTC GCC GGG ATG GAT GTT TTG CCT AGT CAT TGT TGGTTA CGA GAT ATG 244 Val Ala Gly Met Asp Val Leu Pro Ser His Cys Trp LeuArg Asp Met 35 40 45 GTA ACA CAC TTA TCA GTC AGC TTG ACT ACT CTT CTG GACAAG TTT TCA 292 Val Thr His Leu Ser Val Ser Leu Thr Thr Leu Leu Asp LysPhe Ser 50 55 60 AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAACTT GGG 340 Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys LeuGly 65 70 75 80 AAA ATA GTG GAT GAC CTC GTG GCA TGT ATG GAA GAA AAT GCACCT AAG 388 Lys Ile Val Asp Asp Leu Val Ala Cys Met Glu Glu Asn Ala ProLys 85 90 95 AAT GTA AAA GAA TCA CTG AAG AAG CCA GAA ACT AGA AAC TTT ACTCCT 436 Asn Val Lys Glu Ser Leu Lys Lys Pro Glu Thr Arg Asn Phe Thr Pro100 105 110 GAA GAA TTC TTT AGT ATT TTC AAT AGA TCC ATT GAT GCC TTC AAGGAC 484 Glu Glu Phe Phe Ser Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp115 120 125 TTC ATG GTG GCA TCT GAC ACT AGT GAT TGT GTG CTC TCT TCA ACATTA 532 Phe Met Val Ala Ser Asp Thr Ser Asp Cys Val Leu Ser Ser Thr Leu130 135 140 GGT CCT GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATGTTA 580 Gly Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu145 150 155 160 CCC CCT GTT GCA GCC AGT TCC CTT AGG AAT GAC AGC AGT AGCAGT AAT 628 Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser SerAsn 165 170 175 AGG AAA GCC GCA AAG TCC CCT GAA GAC CCA GGC CTA CAA TGGACA GCA 676 Arg Lys Ala Ala Lys Ser Pro Glu Asp Pro Gly Leu Gln Trp ThrAla 180 185 190 ATG GCA CTG CCG GCT CTC ATT TCG CTT GTA ATT GGC TTT GCTTTT GGA 724 Met Ala Leu Pro Ala Leu Ile Ser Leu Val Ile Gly Phe Ala PheGly 195 200 205 GCC TTA TAC TGG AAG AAG AAA CAG TCA AGT CTT ACA AGG GCAGTT GAA 772 Ala Leu Tyr Trp Lys Lys Lys Gln Ser Ser Leu Thr Arg Ala ValGlu 210 215 220 AAT ATA CAG ATT AAT GAA GAG GAT AAT GAG ATA AGT ATG TTGCAA CAG 820 Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu GlnGln 225 230 235 240 AAA GAG AGA GAG TTT CAA GAG GTG TAATT 849 Lys GluArg Glu Phe Gln Glu Val 245 273 amino acids amino acid linear proteinunknown 42 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile Tyr Leu GlnLeu -25 -20 -15 -10 Leu Leu Phe Asn Pro Leu Val Lys Thr Gln Glu Ile CysArg Asn Pro -5 1 5 Val Thr Asp Asn Val Lys Asp Ile Thr Lys Leu Val AlaAsn Leu Pro 10 15 20 Asn Asp Tyr Met Ile Thr Leu Asn Tyr Val Ala Gly MetAsp Val Leu 25 30 35 Pro Ser His Cys Trp Leu Arg Asp Met Val Thr His LeuSer Val Ser 40 45 50 55 Leu Thr Thr Leu Leu Asp Lys Phe Ser Asn Ile SerGlu Gly Leu Ser 60 65 70 Asn Tyr Ser Ile Ile Asp Lys Leu Gly Lys Ile ValAsp Asp Leu Val 75 80 85 Ala Cys Met Glu Glu Asn Ala Pro Lys Asn Val LysGlu Ser Leu Lys 90 95 100 Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu GluPhe Phe Ser Ile Phe 105 110 115 Asn Arg Ser Ile Asp Ala Phe Lys Asp PheMet Val Ala Ser Asp Thr 120 125 130 135 Ser Asp Cys Val Leu Ser Ser ThrLeu Gly Pro Glu Lys Asp Ser Arg 140 145 150 Val Ser Val Thr Lys Pro PheMet Leu Pro Pro Val Ala Ala Ser Ser 155 160 165 Leu Arg Asn Asp Ser SerSer Ser Asn Arg Lys Ala Ala Lys Ser Pro 170 175 180 Glu Asp Pro Gly LeuGln Trp Thr Ala Met Ala Leu Pro Ala Leu Ile 185 190 195 Ser Leu Val IleGly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Lys 200 205 210 215 Gln SerSer Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu 220 225 230 AspAsn Glu Ile Ser Met Leu Gln Gln Lys Glu Arg Glu Phe Gln Glu 235 240 245Val 3807 base pairs nucleic acid single linear protein unknown CDSjoin(905..1018, 1914..1976, 2572..2742, 3152 ..3307, 3512..3597)mat_peptide join(965..1018, 1914..1976, 2572..2742, 3152 ..3307,3512..3597) 43 CACAAGTGAG TAGGGCGCGC CCGGGAGCTC CCAGGCTCTC CAGGAAAAATCGCGCCCGGT 60 GCCCCGGGGA AGCCGGCGCT CCCTGGGACT TGCAGCTGGG GCGTGCAGGGCTGTGCCTGC 120 CGGGTGAGAT ACTACAAAGA TAAATCAGTT GCACAAGTTC TTGAAACTCTACAGTGTAAT 180 AAGGAAAAAT AAGTCATGCA TAAAAGCAAC TATAATACAT AATAGAAAATGTTATTTTCA 240 AGCCGATGTG TAGGTTATGT GTGTTCGAGA GAGAGAGAGA GAAGACAGATTACTTTCTGC 300 TAGGGTTCAA GAATGCCTTC CTGTTGGCTA AGGAAATATT TTCCTTAAGTGGCTAAAAAG 360 CTGTGTTTCA AAATATTCTT TTGATGTCTC ACAAATTCAG TGGAATTCTCTTAGGTCTAA 420 AAATATACAT CTCTCTCACT TTAACTTGGT GTGCTATTGT AGATTATTGGATTAAAGCAC 480 TGCTCAGGGA TTATGCTGCT TCTTGCCAAG CAGTCTACAT TTAAAGTAGAAATAAGATGT 540 TTCTTTTGGT GCCATAAGGT ATACATTTTA TGCATTCTCT AGTTTTTAGAAGATACCCTA 600 AGGGCTAAGT CTTTAACATG CTGCTACAAG TTTATTCCTA ATTGCCATTGGGAAATTGGC 660 TGAAGAAAGT TTTTAACAAA AGTTAACAAT ATTGTCATTG AGAGAATAATTCAAAATGGA 720 TTTTAACTAA AAGCTTTTAA AAACTTTGGT GAGCATAGCT TGAATGCGTAATATTTAATT 780 GCATTTAAGC CAATAACATA TATTAGACTG GTCTTTTTGT GCATCAAGGCATTAGATGTT 840 AAAAGTTTGA ATGATTACAG ATCTTAACTG ATGATCACCA AGCAATTTTTCTGTTTTCAT 900 TTAG ACT TGG ATT CTC ACT TGC ATT TAT CTT CAG CTG CTC CTATTT AAT 949 Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn-20 -15 -10 CCT CTC GTC AAA ACT GAA GGG ATC TGC AGG AAT CGT GTG ACT AATAAT 997 Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn-5 1 5 10 GTA AAA GAC GTC ACT AAA TTG GTAAGTAAGG AATGCTTTAC CGTGCTGTGT1048 Val Lys Asp Val Thr Lys Leu 15 AAAAAAGAGC TGTGGCTCTT TTTCCTGTGCTTGTTGATAA AAGATTTAGA TTTTTCTTGC 1108 CCCAAAGTAA TGTTTTCCTA AAGTGGGGAAAGTAATCACT GGGTTACAAT AAAGGGTTTA 1168 TAGAAAGCAG GTAGTGAGAT ATTTAGGGTCATGGATAATT TGTTGGTAAA ACTGGCTAGT 1228 TGCACACCAC TGCTGTGACT GCTTCTTTGCTGGTCTTCTC CCCATCCTTC ATAGGCAGTG 1288 AAGGACCTTG GAGAGTTCGC TGTGTGCTGATGGGCTTGCC CCAGCTTGTT CCCCATAATC 1348 TCTCCAGTGG GTTTCCCAGC ATGTTCTATTCCCCTTCACA TGTCTTCCTA CTCTTCTTTA 1408 AAAAGCCTAA CGAAAGGAAA TCTGAAATGGCTATTCTCCC AATTCAATCA GCAGGAAGAC 1468 CCTGTCACAT GTCAGTGGGT GTTTGCTCCTTCAGGGAACA TAGAGAGGTG ATTCATTGCC 1528 CACATGTTGA AGGGACTCAT CTCCCTGGTTTGTCACATTG AACTCTTCCC TCAGCGAAAG 1588 CATTTGCATT GCTTCCCGAA TTCCAAGATCACAGGTGGAA GCTGAAATTC AGATCATGTT 1648 TCCAAAACTC AGTAGGTTAT ACCTAGCCAGGCATAACTGA ATTTGGAGTC TAAAAGATCT 1708 GTATTATCAC TTTTTTATTT TGAAGGATGCCTTTTGATTA CAGAGGGAAA TCAAGGATTA 1768 AAAATCAATA TACATGTAAA TATTGAAATTCATTGGTAAC TTTAAAAAGC ACAACAGTTT 1828 TGTGTGCTTT TCTCCAAAGC ACTACAAATATGATTAATTG ATGTATAAGA ATTTTCTTAT 1888 GGAATTTTTT TTTTTGTCTC TGTAG GTGGCA AAT CTT CCA AAA GAC TAC ATG 1940 Val Ala Asn Leu Pro Lys Asp Tyr Met20 25 ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG GTATGTAAAC 1986Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 30 35 TACATTTCTGAGTTTCATTT TAGTAGCTCA TAGAAGAAAT GGGATCATTC ATATTGAGAT 2046 AGTACACTAGCTGCTATTTA GGAGCTTGCT TATTGTCAGG ATTTGAAGAA TTTATCTTTG 2106 GAATTTGACTTGCAGGCTTT TTTTTCCCCC TCTTCCTGTT ACAAGAGTCC CTCCTCCTAT 2166 TACAATAGTCCCTCCTCCTC CTGTCACACT AGTCCCTTCT CTTCCTGTTA CAATAACCCC 2226 TGTCCTCCTATTACAACATT TTAAGTAATG TAATATTAAT TTTAAAAATC TGGCCAGGCA 2286 CGGTGGTTCATGCTTGTAAT CCCAGCACAT TGGGAAGCTG AGACGGGTGG ATCATTTGAG 2346 GTCAGGAAGTTTGAGACAGC CTGGCCAACA TGGTGAAACT TCCTCTCTAC TAAAAATAAA 2406 AAAGTAGCCAGGCATGGTGG CAGGCACTTG TAATCTGAGC TACTCGAGAG GCTGAGGCAG 2466 GAGAATCACTTGAGTAACTA AAACGATAGC TTTGAAGAGT ACTCCGAGTT TTATGGCACT 2526 TACTTATTAAAATAGCTGTT TTGTCTCTTT TTTCATATCT TGCAG CCA AGT CAT 2580 Pro Ser His 40TGT TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT 2628 CysTrp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp 45 50 55 CTTCTG GAC AAG TTT TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC 2676 Leu LeuAsp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser 60 65 70 ATC ATAGAC AAA CTT GTG AAT ATA GTG GAT GAC CTT GTG GAG TGC GTG 2724 Ile Ile AspLys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val 75 80 85 90 AAA GAAAAC TCA TCT AAG GTAACTTTGT GTTCATTGGG ATTATTTTTC 2772 Lys Glu Asn SerSer Lys 95 ATTACGCTTC TCTAAAAACC CATGCTTCTT GGTGCTGTTG GGGAAAATGAGGCACCTTTA 2832 TTTATGATAT TTTGATTGTA TAAACTTCAA ATTTAAAAAT CTTGTTCAGATGAGCAAAGA 2892 AAACAAGTAT TTGCAGTTAT ACTGCAATAC TGAAGTGCAC ATTCTTGTGTTCACTGCCCC 2952 AGATTCAACT TGTGATCCCA CTGGGATCAC TACCCTGCAT TACCAATCTGAATTACATAC 3012 GTTAAAACAG CCATCTAAAA GTGCTAGTTG TAAGAGTCTA AATACTTGAATCTTTGAGAG 3072 ACATATTTAT AGTCCATTAT CTTCACCTCA GTTAAGTCTG AAGACTATTTGAAAAATGTA 3132 ATCCTATTTT TTCTTCTAG GAT CTA AAA AAA TCA TTC AAG AGC CCAGAA CCC 3184 Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro 100 105 AGG CTCTTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT AGA TCC ATT 3232 Arg Leu PheThr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 110 115 120 GAT GCCTTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG 3280 Asp Ala PheLys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val 125 130 135 GTT TCTTCA ACA TTA AGT CCT GAG AAA GGTAAGACAT GTAAGCATTT 3327 Val Ser Ser ThrLeu Ser Pro Glu Lys 140 145 CCAGTTCAAA TGTAAACAAC AAACTTAAAT CTTCCCTATGTAGTAAGAAT CTACCTCTGT 3387 GTTAAGCTGT AGCAAGATAC ATGCATGTAC GTCTAATAAAAAAGCAGATA TCAATAGCAC 3447 AGAAGAAACT CTATAACTCA TACAAATCAC CATATAACACTGACACATTA TTGCTTTCTA 3507 TTTA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTTATG TTA CCC CCT GTT 3556 Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met LeuPro Pro Val 150 155 160 GCA GCC AGC TCC CTT AGG AAT GAC AGC AGT AGC AGTAAT AGTAAGT 3602 Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn 165170 175 ACATATATCT GATTTAATGC ATGCATGGCT CCAATTAGCA CCTATAGGAGTATTGCATGG 3662 GCTTTCAAGG AAACTTCTAC ATTTATTATT ATTGATACTG TTCTGTTACTGTTATTCCTT 3722 TTATGGTCTT CTTGAGACTT AAGTTTGTAG AATTAAATTT CCCTAGAGCTGGAGATAATG 3782 TTTAGAGAAT TAGGCCAATA AATTT 3807 196 amino acids aminoacid linear protein unknown 44 Thr Trp Ile Leu Thr Cys Ile Tyr Leu GlnLeu Leu Leu Phe Asn Pro -20 -15 -10 -5 Leu Val Lys Thr Glu Gly Ile CysArg Asn Arg Val Thr Asn Asn Val 1 5 10 Lys Asp Val Thr Lys Leu Val AlaAsn Leu Pro Lys Asp Tyr Met Ile 15 20 25 Thr Leu Lys Tyr Val Pro Gly MetAsp Val Leu Pro Ser His Cys Trp 30 35 40 Ile Ser Glu Met Val Val Gln LeuSer Asp Ser Leu Thr Asp Leu Leu 45 50 55 60 Asp Lys Phe Ser Asn Ile SerGlu Gly Leu Ser Asn Tyr Ser Ile Ile 65 70 75 Asp Lys Leu Val Asn Ile ValAsp Asp Leu Val Glu Cys Val Lys Glu 80 85 90 Asn Ser Ser Lys Asp Leu LysLys Ser Phe Lys Ser Pro Glu Pro Arg 95 100 105 Leu Phe Thr Pro Glu GluPhe Phe Arg Ile Phe Asn Arg Ser Ile Asp 110 115 120 Ala Phe Lys Asp PheVal Val Ala Ser Glu Thr Ser Asp Cys Val Val 125 130 135 140 Ser Ser ThrLeu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys 145 150 155 Pro PheMet Leu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser 160 165 170 SerSer Ser Asn 175 820 base pairs nucleic acid single linear proteinunknown CDS 17..640 mat_peptide 92..640 45 AAGCTTGCCT TTCCTT ATG AAG AAGACA CAA ACT TGG ATT CTC ACT TGC 49 Met Lys Lys Thr Gln Thr Trp Ile LeuThr Cys -25 -20 -15 ATT TAT CTT CAG CTG CTC CTA TTT AAT CCT CTC GTC AAAACT GAA GGG 97 Ile Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lys ThrGlu Gly -10 -5 1 ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACTAAA TTG 145 Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr LysLeu 5 10 15 GTG GCA AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTCCCC 193 Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro20 25 30 GGG ATG GAT GTT TTG CCA AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA241 Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val 3540 45 50 CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT TCA AAT ATT289 Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile 5560 65 TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA337 Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile 7075 80 GTG GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA385 Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu 8590 95 AAA AAA TCA TTC AAG AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA433 Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu 100105 110 TTC TTT AGA ATT TTT AAT AGA TCC ATT GAT GCC TTC AAG GAC TTT GTA481 Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val 115120 125 130 GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT TCT TCA ACA TTA AGTCCT 529 Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro135 140 145 GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA CCCCCT 577 Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro150 155 160 GTT GCA GCC AGC TCC CTT AGG AAT GAC AGC AGT AGC AGT AAT AGTAAG 625 Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Ser Lys165 170 175 TAC ATA TAT CTG ATT TAATGCATGC ATGGCTCCAA TTAGCACCTATAGGAGTATT 680 Tyr Ile Tyr Leu Ile 180 GCATGGGCTT TCAAGGAAAC TTCTACATTTATTATTATTG ATACTGTTCT GTTACTGTTA 740 TTCCTTTTAT GGTCTTCTTG AGACTTAAGTTTGTAGAATT AAATTTCCCT AGAGCTGGAG 800 ATAATGTTTA GAGAATTAGG 820 208 aminoacids amino acid linear protein unknown 46 Met Lys Lys Thr Gln Thr TrpIle Leu Thr Cys Ile Tyr Leu Gln Leu -25 -20 -15 -10 Leu Leu Phe Asn ProLeu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg -5 1 5 Val Thr Asn Asn ValLys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 10 15 20 Lys Asp Tyr Met IleThr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 25 30 35 Pro Ser His Cys TrpIle Ser Glu Met Val Val Gln Leu Ser Asp Ser 40 45 50 55 Leu Thr Asp LeuLeu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 60 65 70 Asn Tyr Ser IleIle Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 75 80 85 Glu Cys Val LysGlu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 90 95 100 Ser Pro GluPro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe 105 110 115 Asn ArgSer Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr 120 125 130 135Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg 140 145150 Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser 155160 165 Leu Arg Asn Asp Ser Ser Ser Ser Asn Ser Lys Tyr Ile Tyr Leu Ile170 175 180 5864 base pairs nucleic acid single linear DNA unknown CDSjoin(565..579, 1684..1797, 2693..2755, 3351..3521, 3932..4088,4314..4397, 4778..4887, 5208..5275, 5677..5713) mat_peptidejoin(1744..1797, 2693..2755, 3351..3521, 3932..4088, 4314..4397,4778..4887, 5208..5275, 5677..5713) 47 GAGCTCCGAG CCCTCTCTGG CGCGCGAGGTATTTCGTCTG TNCCCGGGGG TGCCAGGTGA 60 GCCCCAGCGG ATCCGGGAGG GTAAGCTGGGACTCCTCGCG AGCAGTAGCT GCAGGGTACC 120 AAGCTTCGCC CTCTGCGTCC CCGCGCCTTCGCGGTCTCCC GCCAGTGCAG GTCCGGGGCC 180 CCCAGGCGAG CGGACAAGGT TGGCCTAATCTGCCAAACTT CTGGGGCATT TACCGTGCTC 240 TGGCCGCCCT CCCGATTCTT CCCTCCGCGCCCTTGCCTGC TTCTCGCCTA CCCCGGGCTC 300 CGGAAGGGAA GGAGGCGTGT CCGGAGCAGGCGGGCGGGAA CTGTATAAAA GCGCCGGCGG 360 CTCAGCAGCC GGCTTCGCTC GCCGCCTCGCGCCGAGACTA GAAGCGCTGC GGGAAGCAGG 420 GACAGTGGAG AGGGCGCTGC GCTCGGGCTACCCAATGCGT GGACTATCTG CCGCCGCTGT 480 TCGTGCAATA TGCTGGAGCT CCAGAACAGCTAAACGGAGT CGCCACACCA CTGTTTGTGC 540 TGGATCGCAC CGCTGCCTTT CCTT ATG AAGAAG ACA CAA GTGAGTAGGG 589 Met Lys Lys Thr Gln -25 CGCGCCCGGG AGCTCCCAGGCTCTCCAGGA AAAATCGCGC CCGGTGCCCC GGGGAAGCCG 649 GCGCTCCCTG GGACTTGCAGCTGGGGCGTG CAGGGCTGTG CCTGCCGGGT GAGACAAGAG 709 GATGCGGGGG AGGCCGGCGTGGTGTGTGAT CCCGAGCCGA GCCGNNTGAG CCAGGGAGAA 769 AAGGAGTGGG AGTACTGAGAGGGAGCCAGT GTCAAGTTTG GAGCCTCAGC AGTTAAGTTT 829 TGAGCTGTCA GTCGGAAACCGTAATTCCCG TCTGGTGGAA AGATTGGCTT TTNGNCCACG 889 GAATGTAAGT TATCACAGATACTACAAAGA TAAATCAGTT GCACAAGTTC TTGAAACTCT 949 ACAGTGTAAT AAGGAAAAATAAGTCATGCA TAAAAGCAAC TATAATACAT AATAGAAAAT 1009 GTTATTTTCA AGCCGATGTGTAGGTTATGT GTGTTCGAGA GAGAGAGAGA GAAGACAGAT 1069 TACTTTCTGC TAGGGTTCAAGAATGCCTTC CTGTTGGCTA AGGAAATATT TTCCTTAAGT 1129 GGCTAAAAAG CTGTGTTTCAAAATATTCTT TTGATGTCTC ACAAATTCAG TGGAATTCTC 1189 TTAGGTCTAA AAATATACATCTCTCTCACT TTAACTTGGT GTGCTATTGT AGATTATTGG 1249 ATTAAAGCAC TGCTCAGGGATTATGCTGCT TCTTGCCAAG CAGTCTACAT TTAAAGTAGA 1309 AATAAGATGT TTCTTTTGGTGCCATAAGGT ATACATTTTA TGCATTCTCT AGTTTTTAGA 1369 AGATACCCTA AGGGCTAAGTCTTTAACATG CTGCTACAAG TTTATTCCTA ATTGCCATTG 1429 GGAAATTGGC TGAAGAAAGTTTTTAACAAA AGTTAACAAT ATTGTCATTG AGAGAATAAT 1489 TCAAAATGGA TTTTAACTAAAAGCTTTTAA AAACTTTGGT GAGCATAGCT TGAATGCGTA 1549 ATATTTAATT GCATTTAAGCCAATAACATA TATTAGACTG GTCTTTTTGT GCATCAAGGC 1609 ATTAGATGTT AAAAGTTTGAATGATTACAG ATCTTAACTG ATGATCACCA AGCAATTTTT 1669 CTGTTTTCAT TTAG ACT TGGATT CTC ACT TGC ATT TAT CTT CAG CTG CTC 1719 Thr Trp Ile Leu Thr Cys IleTyr Leu Gln Leu Leu -20 -15 -10 CTA TTT AAT CCT CTC GTC AAA ACT GAA GGGATC TGC AGG AAT CGT GTG 1767 Leu Phe Asn Pro Leu Val Lys Thr Glu Gly IleCys Arg Asn Arg Val -5 1 5 ACT AAT AAT GTA AAA GAC GTC ACT AAA TTGGTAAGTAAGG AATGCTTTAC 1817 Thr Asn Asn Val Lys Asp Val Thr Lys Leu 10 15CGTGCTGTGT AAAAAAGAGC TGTGGCTCTT TTTCCTGTGC TTGTTGATAA AAGATTTAGA 1877TTTTTCTTGC CCCAAAGTAA TGTTTTCCTA AAGTGGGGAA AGTAATCACT GGGTTACAAT 1937AAAGGGTTTA TAGAAAGCAG GTAGTGAGAT ATTTAGGGTC ATGGATAATT TGTTGGTAAA 1997ACTGGCTAGT TGCACACCAC TGCTGTGACT GCTTCTTTGC TGGTCTTCTC CCCATCCTTC 2057ATAGGCAGTG AAGGACCTTG GAGAGTTCGC TGTGTGCTGA TGGGCTTGCC CCAGCTTGTT 2117CCCCATAATC TCTCCAGTGG GTTTCCCAGC ATGTTCTATT CCCCTTCACA TGTCTTCCTA 2177CTCTTCTTTA AAAAGCCTAA CGAAAGGAAA TCTGAAATGG CTATTCTCCC AATTCAATCA 2237GCAGGAAGAC CCTGTCACAT GTCAGTGGGT GTTTGCTCCT TCAGGGAACA TAGAGAGGTG 2297ATTCATTGCC CACATGTTGA AGGGACTCAT CTCCCTGGTT TGTCACATTG AACTCTTCCC 2357TCAGCGAAAG CATTTGCATT GCTTCCCGAA TTCCAAGATC ACAGGTGGAA GCTGAAATTC 2417AGATCATGTT TCCAAAACTC AGTAGGTTAT ACCTAGCCAG GCATAACTGA ATTTGGAGTC 2477TAAAAGATCT GTATTATCAC TTTTTTATTT TGAAGGATGC CTTTTGATTA CAGAGGGAAA 2537TCAAGGATTA AAAATCAATA TACATGTAAA TATTGAAATT CATTGGTAAC TTTAAAAAGC 2597ACAACAGTTT TGTGTGCTTT TCTCCAAAGC ACTACAAATA TGATTAATTG ATGTATAAGA 2657ATTTTCTTAT GGAATTTTTT TTTTTGTCTC TGTAG GTG GCA AAT CTT CCA AAA 2710 ValAla Asn Leu Pro Lys 20 GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATGGAT GTT TTG 2755 Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp ValLeu 25 30 35 GTATGTAAAC TACATTTCTG AGTTTCATTT TAGTAGCTCA TAGAAGAAATGGGATCATTC 2815 ATATTGAGAT AGTACACTAG CTGCTATTTA GGAGCTTGCT TATTGTCAGGATTTGAAGAA 2875 TTTATCTTTG GAATTTGACT TGCAGGCTTT TTTTTCCCCC TCTTCCTGTTACAAGAGTCC 2935 CTCCTCCTAT TACAATAGTC CCTCCTCCTC CTGTCACACT AGTCCCTTCTCTTCCTGTTA 2995 CAATAACCCC TGTCCTCCTA TTACAACATT TTAAGTAATG TAATATTAATTTTAAAAATC 3055 TGGCCAGGCA CGGTGGTTCA TGCTTGTAAT CCCAGCACAT TGGGAAGCTGAGACGGGTGG 3115 ATCATTTGAG GTCAGGAAGT TTGAGACAGC CTGGCCAACA TGGTGAAACTTCCTCTCTAC 3175 TAAAAATAAA AAAGTAGCCA GGCATGGTGG CAGGCACTTG TAATCTGAGCTACTCGAGAG 3235 GCTGAGGCAG GAGAATCACT TGAGTAACTA AAACGATAGC TTTGAAGAGTACTCCGAGTT 3295 TTATGGCACT TACTTATTAA AATAGCTGTT TTGTCTCTTT TTTCATATCTTGCAG CCA 3353 Pro 40 AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA CAA TTGTCA GAC AGC TTG 3401 Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu SerAsp Ser Leu 45 50 55 ACT GAT CTT CTG GAC AAG TTT TCA AAT ATT TCT GAA GGCTTG AGT AAT 3449 Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly LeuSer Asn 60 65 70 TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG GAT GAC CTTGTG GAG 3497 Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu ValGlu 75 80 85 TGC GTG AAA GAA AAC TCA TCT AAG GTAACTTTGT GTTCATTGGGATTATTTTTC 3551 Cys Val Lys Glu Asn Ser Ser Lys 90 95 ATTACGCTTCTCTAAAAACC CATGCTTCTT GGTGCTGTTG GGGAAAATGA GGCACCTTTA 3611 TTTATGATATTTTGATTGTA TAAACTTCAA ATTTAAAAAT CTTGTTCAGA TGAGCAAAGA 3671 AAACAAGTATTTGCAGTTAT ACTGCAATAC TGAAGTGCAC TATTCTTGTG TTCACTGCCC 3731 CAGATTCAACTTGTGATCCC ACTGGGATCA CTACCCTGCA TTACCAATCT GAATTACATA 3791 CGTTAAAACAGCCATCTAAA AGTGCTAGTT GTAAGAGTCT AAATACTTGA ATCTTTGAGA 3851 GACATATTTATAGTCCATTA TCTTCACCTC AGTTAAGTCT GAAGACTATT TGAAAAATGT 3911 AATCCTATTTTTTCTTCTAG GAT CTA AAA AAA TCA TTC AAG AGC CCA GAA 3961 Asp Leu Lys LysSer Phe Lys Ser Pro Glu 100 105 CCC AGG CTC TTT ACT CCT GAA GAA TTC TTTAGA ATT TTT AAT AGA TCC 4009 Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe ArgIle Phe Asn Arg Ser 110 115 120 ATT GAT GCC TTC AAG GAC TTT GTA GTG GCATCT GAA ACT AGT GAT TGT 4057 Ile Asp Ala Phe Lys Asp Phe Val Val Ala SerGlu Thr Ser Asp Cys 125 130 135 GTG GTT TCT TCA ACA TTA AGT CCT GAG AAAG GTAAGACATG TAAGCATTTC 4108 Val Val Ser Ser Thr Leu Ser Pro Glu Lys 140145 CAGTTCAAAT GTAAACAACA AACTTAAATC TTCCCTATGT AGTAAGAATC TACCTCTGTG4168 TTAAGCTGTA GCAAGATACA TGCATGTACG TCTAAAAAAA AGCAGATATC AATAGCACAG4228 AAGAAACTAA TGATTGTAGA TTTGTGGGCT CTATAACTCA TACAAATCAC CATATAACAC4288 TGACACATTA TTGCTTTCTA TTTAG AT TCC AGA GTC AGT GTC ACA AAA CCA 4339Asp Ser Arg Val Ser Val Thr Lys Pro 150 155 TTT ATG TTA CCC CCT GTT GCAGCC AGC TCC CTT AGG AAT GAC AGC AGT 4387 Phe Met Leu Pro Pro Val Ala AlaSer Ser Leu Arg Asn Asp Ser Ser 160 165 170 AGC AGT AAT A GTAAGTACATATATCTGATT TAATGCATGC ATGGCTCCAA 4437 Ser Ser Asn 175 TTAGCACCTATAGGAGTATT GCATGGGCTT TCAAGGAAAC TTCTACATTT ATTATTATTG 4497 ATACTGTTCTGTTACTGTTA TTCCTTTTAT GGTCTTCTTG AGACTTAAGT TTGTAGAATT 4557 AAATTTCCCTAGAGCTGGAG ATAATGTTTA GAGAATTAGG CCAATAAATT TTCTGCTGAG 4617 GTTATTTTAAATAAGACATA AAATTAATTT TAGAAATATG ATTTATGCCT TTTGTTGAAT 4677 CATTAACATATATACAGAAA CAGTTAAAAC AACCACAGCA TAAGAGAAAA ACTTCTAGAA 4737 TGGATATGCTGTATTCATCA GTGTGTTCTT TAAATTATAG GG AAG GCC AAA AAT 4791 Arg Lys Ala LysAsn 180 CCC CCT GGA GAC TCC AGC CTA CAC TGG GCA GCC ATG GCA TTG CCA GCA4839 Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala 185190 195 TTG TTT TCT CTT ATA ATT GGC TTT GCT TTT GGA GCC TTA TAC TGG AAG4887 Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys 200205 210 GTAAGTGGTA CCATTCCTTT TTTTAAAAAT ATGCTATGTT TACATAAATTATCATCTTTT 4947 TTTCCTCAAG AAATGATCCT TTAAGAAAAC AGTGAATCTA CCTTAGCTTATACTAAACAA 5007 AATTTAAATT TTATAAAGTT TCCTGTTTCT CATTATGTCT GGAGACAATCCCTCTAGCTG 5067 ATAATTCACG CTTAAGAATT AGGAACTAAA ACTGTTATTG GAGTTATTGCCATAAAAGAT 5127 AAAAGTGGAG TCCACTTACC TCTTAAATAT TAGACCATTC ATTGATTATTTTACAGTATA 5187 TGTCTTTCTT CTTTTTCCAG AAG AGA CAG CCA AGT CTT ACA AGGGCA GTT 5237 Lys Arg Gln Pro Ser Leu Thr Arg Ala Val 215 220 GAA AAT ATACAA ATT AAT GAA GAG GAT AAT GAG ATA AG GTATTTTGTT 5285 Glu Asn Ile GlnIle Asn Glu Glu Asp Asn Glu Ile Ser 225 230 235 TTGCTAAATG TGTGCCCAATCAAGCATGAC ATTGCCATTT CACACACTGT GTACCTGCCC 5345 ATAATGTCTT TAAGAAGTCCTTCACTCATG ACAGTAGCTC CTAACCAGTG AGTCCCAACT 5405 CTATCCATGT TTCTGATGTCTCACTCTCTC TTCGTATGTG TATATGCATA TACAGAGAAA 5465 GAAATGTTTT AACTACTTGGAAAGACTACC TTAAGACAAA TGAAGTCTTC CCTCTTCCCT 5525 ATAGTAATAA GAAGGTAGGCTCCCCCATTC AATTTTGCAA TCTTCTGCTA CTATATTTAC 5585 AGAAAAGCTG CCTTTTACAATGCCGAGATC ATGGTGTACC TCAGAATCTC TGACCAAGAG 5645 CAAATAAGCA TTTTTTCTTATTGTTTTTCA G T ATG TTG CAA GAG AAA GAG AGA 5698 Met Leu Gln Glu Lys GluArg 240 GAG TTT CAA GAA GTG TAATTGTGGC TTGTATCAAC ACTGTTACTT TCGTACATTG5753 Glu Phe Gln Glu Val 245 GTAAGTTTTT TTCTTCTTTC CTTTTTTTTT CTTTTTTTTATTATACTTTA AGTTCTAGGG 5813 TACATGTGCA CAATGTGCAG GTTTGTTACG TATGTTTACATGTGCCATGT T 5864 273 amino acids amino acid linear protein unknown 48Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu -25 -20-15 -10 Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg-5 1 5 Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro10 15 20 Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu25 30 35 Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser40 45 50 55 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly LeuSer 60 65 70 Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp LeuVal 75 80 85 Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser PheLys 90 95 100 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe ArgIle Phe 105 110 115 Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val AlaSer Glu Thr 120 125 130 135 Ser Asp Cys Val Val Ser Ser Thr Leu Ser ProGlu Lys Asp Ser Arg 140 145 150 Val Ser Val Thr Lys Pro Phe Met Leu ProPro Val Ala Ala Ser Ser 155 160 165 Leu Arg Asn Asp Ser Ser Ser Ser AsnArg Lys Ala Lys Asn Pro Pro 170 175 180 Gly Asp Ser Ser Leu His Trp AlaAla Met Ala Leu Pro Ala Leu Phe 185 190 195 Ser Leu Ile Ile Gly Phe AlaPhe Gly Ala Leu Tyr Trp Lys Lys Arg 200 205 210 215 Gln Pro Ser Leu ThrArg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu 220 225 230 Asp Asn Glu IleSer Met Leu Gln Glu Lys Glu Arg Glu Phe Gln Glu 235 240 245 Val 273amino acids amino acid single linear protein unknown 49 Met Lys Lys ThrGln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu 1 5 10 15 Leu Leu PheAsn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg 20 25 30 Val Thr AsnAsn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 35 40 45 Lys Asp TyrMet Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 50 55 60 Pro Ser HisCys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser 65 70 75 80 Leu ThrAsp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95 Asn TyrSer Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 100 105 110 GluCys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 115 120 125Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe 130 135140 Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr 145150 155 160 Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp SerArg 165 170 175 Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala AlaSer Ser 180 185 190 Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala LysAsn Pro Pro 195 200 205 Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala LeuPro Ala Leu Phe 210 215 220 Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala LeuTyr Trp Lys Lys Arg 225 230 235 240 Gln Pro Ser Leu Thr Arg Ala Val GluAsn Ile Gln Ile Asn Glu Glu 245 250 255 Asp Asn Glu Ile Ser Met Leu GlnGlu Lys Glu Arg Glu Phe Gln Glu 260 265 270 Val 273 amino acids aminoacid single linear protein unknown 50 Met Lys Lys Thr Gln Thr Trp IleLeu Thr Cys Ile Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu ValLys Thr Glu Gly Ile Cys Arg Asn Arg 20 25 30 Val Thr Asn Asn Val Lys AspVal Thr Lys Leu Val Ala Asn Leu Pro 35 40 45 Lys Asp Tyr Met Ile Thr LeuLys Tyr Val Pro Gly Met Asp Val Leu 50 55 60 Pro Ser His Cys Trp Ile SerGlu Met Val Val Gln Leu Ser Asp Ser 65 70 75 80 Leu Thr Asp Leu Leu AspLys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95 Asn Tyr Ser Ile Ile AspLys Leu Val Asn Ile Val Asp Asp Leu Val 100 105 110 Glu Cys Val Lys GluAsn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 115 120 125 Ser Pro Glu ProArg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe 130 135 140 Asn Arg SerIle Asp Ala Phe Lys Asp Phe Ala Val Ala Ser Glu Thr 145 150 155 160 SerAsp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg 165 170 175Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser 180 185190 Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Lys Asn Pro Thr 195200 205 Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala Phe Phe210 215 220 Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys LysArg 225 230 235 240 Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln IleAsn Glu Asp 245 250 255 Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu ArgGlu Phe Gln Glu 260 265 270 Val 274 amino acids amino acid single linearprotein unknown 51 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile TyrLeu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu Val Lys Thr Lys Gly IleCys Gly Lys Arg 20 25 30 Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu ValAla Asn Leu Pro 35 40 45 Lys Asp Tyr Lys Ile Ala Leu Lys Tyr Val Pro GlyMet Asp Val Leu 50 55 60 Pro Ser His Cys Trp Ile Ser Val Met Val Glu GlnLeu Ser Val Ser 65 70 75 80 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn IleSer Glu Gly Leu Ser 85 90 95 Asn Tyr Ser Ile Ile Asp Lys Leu Val Lys IleVal Asp Asp Leu Val 100 105 110 Glu Cys Thr Glu Gly Tyr Ser Phe Glu AsnVal Lys Lys Ala Pro Lys 115 120 125 Ser Pro Glu Leu Arg Leu Phe Thr ProGlu Glu Phe Phe Arg Ile Phe 130 135 140 Asn Arg Ser Ile Asp Ala Phe LysAsp Leu Glu Thr Val Ala Ser Lys 145 150 155 160 Ser Ser Glu Cys Val ValSer Ser Thr Leu Ser Pro Asp Lys Asp Ser 165 170 175 Arg Val Ser Val ThrLys Pro Phe Met Leu Pro Pro Val Ala Ala Ser 180 185 190 Ser Leu Arg AsnAsp Ser Ser Ser Ser Asn Arg Lys Ala Ser Asn Ser 195 200 205 Ile Gly AspSer Asn Leu Gln Trp Ala Ala Met Ala Leu Pro Ala Phe 210 215 220 Phe SerLeu Val Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys 225 230 235 240Lys Gln Pro Asn Leu Thr Arg Thr Val Glu Asn Ile Gln Ile Asn Glu 245 250255 Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu Phe Gln 260265 270 Glu Val 274 amino acids amino acid single linear protein unknown52 Met Lys Xaa Thr Gln Thr Trp Ile Val Thr Cys Ile Tyr Leu Gln Xaa 1 510 15 Leu Leu Phe Asn Pro Leu Val Lys Thr Lys Gly Leu Cys Arg Asn Arg 2025 30 Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 3540 45 Lys Asp Tyr Lys Ile Ala Leu Lys Tyr Val Pro Gly Met Asp Val Leu 5055 60 Pro Ser His Cys Trp Ile Ser Val Met Val Glu Gln Leu Ser Val Ser 6570 75 80 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser85 90 95 Asn Tyr Ser Ile Ile Asp Lys Leu Val Lys Ile Val Asp Asp Leu Val100 105 110 Glu Cys Val Glu Gly His Ser Ser Glu Asn Val Lys Lys Ser SerLys 115 120 125 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe ArgIle Phe 130 135 140 Asn Arg Ser Ile Asp Ala Phe Lys Asp Leu Glu Met ValAla Ser Lys 145 150 155 160 Thr Ser Glu Cys Val Val Ser Ser Thr Leu SerPro Glu Lys Asp Ser 165 170 175 Arg Val Ser Val Thr Lys Pro Phe Met LeuPro Pro Val Ala Ala Ser 180 185 190 Ser Leu Arg Asn Asp Ser Ser Ser SerAsn Arg Lys Xaa Thr Asn Pro 195 200 205 Ile Glu Asp Ser Ser Ile Gln TrpAla Val Met Ala Leu Pro Ala Cys 210 215 220 Phe Ser Leu Val Ile Gly PheAla Phe Gly Ala Phe Tyr Trp Lys Lys 225 230 235 240 Lys Gln Pro Asn LeuThr Arg Thr Val Glu Asn Ile Gln Ile Asn Glu 245 250 255 Glu Asp Asn GluIle Ser Met Leu Gln Glu Lys Glu Arg Glu Phe Gln 260 265 270 Glu Val 274amino acids amino acid single linear protein unknown 53 Met Lys Lys ThrGln Thr Trp Ile Ile Thr Cys Ile Tyr Leu Gln Leu 1 5 10 15 Leu Leu PheAsn Pro Leu Val His Thr Gln Gly Ile Cys Ser Asn Arg 20 25 30 Val Thr AspAsp Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 35 40 45 Lys Asp TyrMet Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 50 55 60 Pro Ser HisCys Trp Ile Ser Glu Met Val Glu Gln Leu Ser Val Ser 65 70 75 80 Leu ThrAsp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95 Asn TyrCys Ile Ile Asp Lys Leu Val Lys Ile Val Asp Asp Leu Val 100 105 110 GluCys Met Glu Xaa His Ser Ser Glu Asn Val Lys Lys Ser Ser Lys 115 120 125Ser Pro Glu Pro Arg Gln Phe Thr Pro Glu Lys Phe Phe Gly Ile Phe 130 135140 Asn Lys Ser Ile Asp Ala Phe Lys Asp Leu Glu Ile Val Ala Ser Lys 145150 155 160 Met Ser Glu Cys Val Ile Ser Ser Thr Ser Ser Pro Glu Lys AspSer 165 170 175 Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val AlaAla Ser 180 185 190 Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys AlaSer Asn Ser 195 200 205 Ile Glu Asp Ser Ser Leu Gln Trp Ala Ala Val AlaLeu Pro Ala Phe 210 215 220 Phe Ser Leu Val Ile Gly Phe Ala Phe Gly AlaPhe Tyr Trp Lys Lys 225 230 235 240 Lys Gln Pro Asn Leu Thr Arg Thr ValGlu Asn Arg Gln Ile Asn Glu 245 250 255 Glu Asp Asn Glu Ile Ser Met LeuGln Glu Lys Glu Arg Glu Phe Gln 260 265 270 Glu Val 273 amino acidsamino acid single linear protein unknown 54 Met Lys Lys Thr Gln Thr TrpIle Ile Thr Cys Ile Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro LeuVal Lys Thr Gln Glu Ile Cys Arg Asn Pro 20 25 30 Val Thr Asp Asn Val LysAsp Ile Thr Lys Leu Val Ala Asn Leu Pro 35 40 45 Asn Asp Tyr Met Ile ThrLeu Asn Tyr Val Ala Gly Met Asp Val Leu 50 55 60 Pro Ser His Cys Trp LeuArg Asp Met Val Thr His Leu Ser Val Ser 65 70 75 80 Leu Thr Thr Leu LeuAsp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95 Asn Tyr Ser Ile IleAsp Lys Leu Gly Lys Ile Val Asp Asp Leu Val 100 105 110 Ala Cys Met GluGlu Asn Ala Pro Lys Asn Val Lys Glu Ser Leu Lys 115 120 125 Lys Pro GluThr Arg Asn Phe Thr Pro Glu Glu Phe Phe Ser Ile Phe 130 135 140 Asn ArgSer Ile Asp Ala Phe Lys Asp Phe Met Val Ala Ser Asp Thr 145 150 155 160Ser Asp Cys Val Leu Ser Ser Thr Leu Gly Pro Glu Lys Asp Ser Arg 165 170175 Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser 180185 190 Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Ala Lys Ser Pro195 200 205 Glu Asp Pro Gly Leu Gln Trp Thr Ala Met Ala Leu Pro Ala LeuIle 210 215 220 Ser Leu Val Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp LysLys Lys 225 230 235 240 Gln Ser Ser Leu Thr Arg Ala Val Glu Asn Ile GlnIle Asn Glu Glu 245 250 255 Asp Asn Glu Ile Ser Met Leu Gln Gln Lys GluArg Glu Phe Gln Glu 260 265 270 Val 273 amino acids amino acid singlelinear protein unknown 55 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr CysIle Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu Val Lys Thr LysGlu Ile Cys Gly Asn Pro 20 25 30 Val Thr Asp Asn Val Lys Asp Ile Thr LysLeu Val Ala Asn Leu Pro 35 40 45 Asn Asp Tyr Met Ile Thr Leu Asn Tyr ValAla Gly Met Asp Val Leu 50 55 60 Pro Ser His Cys Trp Leu Arg Asp Met ValIle Gln Leu Ser Leu Ser 65 70 75 80 Leu Thr Thr Leu Leu Asp Lys Phe SerAsn Ile Ser Glu Gly Leu Ser 85 90 95 Asn Tyr Ser Ile Ile Asp Lys Leu GlyLys Ile Val Asp Asp Leu Val 100 105 110 Leu Cys Met Glu Glu Asn Ala ProLys Asn Ile Lys Glu Ser Pro Lys 115 120 125 Arg Pro Glu Thr Arg Ser PheThr Pro Glu Glu Phe Phe Ser Ile Phe 130 135 140 Asn Arg Ser Ile Asp AlaPhe Lys Asp Phe Met Val Ala Ser Asp Thr 145 150 155 160 Ser Asp Cys ValLeu Ser Ser Thr Leu Gly Pro Glu Lys Asp Ser Arg 165 170 175 Val Ser ValThr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser 180 185 190 Leu ArgAsn Asp Ser Ser Ser Ser Asn Arg Lys Ala Ala Lys Ala Pro 195 200 205 GluAsp Ser Gly Leu Gln Trp Thr Ala Met Ala Leu Pro Ala Leu Ile 210 215 220Ser Leu Val Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Lys 225 230235 240 Gln Ser Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu245 250 255 Asp Asn Glu Ile Ser Met Leu Gln Gln Lys Glu Arg Glu Phe GlnGlu 260 265 270 Val 282 amino acids amino acid single linear proteinunknown 56 Thr Trp Ile Ile Thr Cys Phe Cys Leu Gln Leu Leu Leu Leu AsnPro 1 5 10 15 Leu Val Lys Ala Gln Ser Ser Cys Gly Asn Pro Val Thr AspAsp Val 20 25 30 Asn Asp Ile Ala Lys Leu Val Gly Asn Leu Pro Asn Asp TyrLeu Ile 35 40 45 Thr Leu Lys Tyr Val Pro Lys Met Asp Ser Leu Pro Asn HisCys Trp 50 55 60 Leu His Leu Met Val Pro Glu Phe Ser Arg Ser Leu His AsnLeu Leu 65 70 75 80 Gln Lys Phe Ser Asp Ile Ser Asp Met Ser Asp Val LeuSer Asn Tyr 85 90 95 Ser Ile Ile Asn Asn Leu Thr Arg Ile Ile Asn Asp LeuMet Ala Cys 100 105 110 Leu Ala Phe Asp Lys Asn Lys Asp Phe Ile Lys GluAsn Gly His Leu 115 120 125 Tyr Glu Glu Asp Arg Phe Ile Pro Glu Asn PhePhe Arg Leu Phe Asn 130 135 140 Ser Thr Ile Glu Val Tyr Lys Glu Phe AlaAsp Ser Leu Asp Lys Asn 145 150 155 160 Asp Cys Ile Met Pro Ser Thr ValGlu Thr Pro Glu Asn Asp Ser Arg 165 170 175 Val Ala Val Thr Lys Thr IleSer Phe Pro Pro Val Ala Ala Ser Ser 180 185 190 Leu Arg Asn Asp Ser IleGly Ser Asn Thr Ser Ser Asn Ser Asn Lys 195 200 205 Glu Ala Leu Gly PheIle Ser Ser Ser Ser Leu Gln Gly Ile Ser Ile 210 215 220 Ala Leu Thr SerLeu Leu Ser Leu Leu Ile Gly Phe Ile Leu Gly Ala 225 230 235 240 Ile TyrTrp Lys Lys Thr His Pro Lys Ser Arg Pro Glu Ser Asn Glu 245 250 255 ThrIle Gln Cys His Gly Cys Gln Glu Glu Asn Glu Ile Ser Met Leu 260 265 270Gln Gln Lys Glu Lys Glu His Leu Gln Val 275 280 266 amino acids aminoacid single linear protein unknown 57 Met Lys Lys Thr Gln Thr Trp IleIle Thr Cys Ile Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu ValLys Thr Gly Ile Cys Arg Asn Arg Val 20 25 30 Thr Asp Val Lys Asp Val ThrLys Leu Val Ala Asn Leu Pro Lys Asp 35 40 45 Tyr Met Ile Thr Leu Lys TyrVal Pro Gly Met Asp Val Leu Pro Ser 50 55 60 His Cys Trp Ile Ser Glu MetVal Glu Gln Leu Ser Val Ser Leu Thr 65 70 75 80 Asp Leu Leu Asp Lys PheSer Asn Ile Ser Glu Gly Leu Ser Asn Tyr 85 90 95 Ser Ile Ile Asp Lys LeuVal Lys Ile Val Asp Asp Leu Val Glu Cys 100 105 110 Glu Glu Asn Ser SerLys Asn Val Lys Lys Ser Lys Ser Pro Glu Pro 115 120 125 Arg Leu Phe ThrPro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile 130 135 140 Asp Ala PheLys Asp Phe Met Val Ala Ser Lys Thr Ser Asp Cys Val 145 150 155 160 ValSer Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr 165 170 175Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp 180 185190 Ser Ser Ser Ser Asn Arg Lys Ala Asn Glu Asp Ser Ser Leu Gln Trp 195200 205 Ala Ala Met Ala Leu Pro Ala Leu Phe Ser Leu Val Ile Gly Phe Ala210 215 220 Phe Gly Ala Leu Tyr Trp Lys Lys Lys Gln Pro Ser Leu Thr ArgAla 225 230 235 240 Val Glu Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu IleSer Met Leu 245 250 255 Gln Glu Lys Glu Arg Glu Phe Gln Glu Val 260 265269 base pairs nucleic acid single linear protein unknown CDSjoin(1..210, 223..258) 58 GAA TTC TTC CGT ATC TTC AAC CGT TCC ATC GACGCT TTC AAA GAC TTC 48 Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp AlaPhe Lys Asp Phe 1 5 10 15 GTT GTT GCT TCC GAA ACC TCC GAC TGC GTT GTTTCC TCC ACC CTG TCT 96 Val Val Ala Ser Glu Thr Ser Asp Cys Val Val SerSer Thr Leu Ser 20 25 30 CCG GAA AAA GAC TCC CGT GTT TCG GTT ACC AAA CCGTTC ATG CTG CCG 144 Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro PheMet Leu Pro 35 40 45 CCG GTT GCT GCT TCC TCC CTG CGT AAC GAC TCC TCC TCCTCC AAC TCC 192 Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser SerAsn Ser 50 55 60 AAA TAC ATC TAC CTG ATC TAATAGGATC CG GTT ACC AAA CCGTTC ATG 240 Lys Tyr Ile Tyr Leu Ile Val Thr Lys Pro Phe Met 65 70 75 CTGCCG CCG GTT GCT GCT TAATAGGATC C 269 Leu Pro Pro Val Ala Ala 80 82 aminoacids amino acid linear protein unknown 59 Glu Phe Phe Arg Ile Phe AsnArg Ser Ile Asp Ala Phe Lys Asp Phe 1 5 10 15 Val Val Ala Ser Glu ThrSer Asp Cys Val Val Ser Ser Thr Leu Ser 20 25 30 Pro Glu Lys Asp Ser ArgVal Ser Val Thr Lys Pro Phe Met Leu Pro 35 40 45 Pro Val Ala Ala Ser SerLeu Arg Asn Asp Ser Ser Ser Ser Asn Ser 50 55 60 Lys Tyr Ile Tyr Leu IleVal Thr Lys Pro Phe Met Leu Pro Pro Val 65 70 75 80 Ala Ala 1404 basepairs nucleic acid single linear protein unknown CDS 184..1002mat_peptide 259..1002 60 CCGCCTCGCG CCGAGACTAG AAGCGCTGCG GGAAGCAGGGACAGTGGAGA GGGCGCTGCG 60 CTCGGGCTAC CCAATGCGTG GACTATCTGC CGCCGCTGTTCGTGCAATAT GCTGGAGCTC 120 CAGAACAGCT AAACGGAGTC GCCACACCAC TGTTTGTGCTGGATCGCAGC GCTGCCTTTC 180 CTT ATG AAG AAG ACA CAA ACT TGG ATT CTC ACTTGC ATT TAT CTT CAG 228 Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys IleTyr Leu Gln -25 -20 -15 CTG CTC CTA TTT AAT CCT CTC GTC AAA ACT GAA GGGATC TGC AGG AAT 276 Leu Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly IleCys Arg Asn -10 -5 1 5 CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTGGTG GCA AAT CTT 324 Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu ValAla Asn Leu 10 15 20 CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGGATG GAT GTT 372 Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly MetAsp Val 25 30 35 TTG CCA AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA CAA TTGTCA GAC 420 Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu SerAsp 40 45 50 AGC TTG ACT GAT CTT CTG GAC AAG TTT TCA AAT ATT TCT GAA GGCTTG 468 Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu55 60 65 70 AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG GAT GACCTT 516 Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu75 80 85 GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA TCA TTC564 Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe 9095 100 AAG AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT612 Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile 105110 115 TTT AAT AGA TCC ATT GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA660 Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu 120125 130 ACT AGT GAT TGT GTG GTT TCT TCA ACA TTA AGT CCT GAG AAA GAT TCC708 Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser 135140 145 150 AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA CCC CCT GTT GCA GCCAGC 756 Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser155 160 165 TCC CTT AGG AAT GAC AGC AGT AGC AGT AAT AGG AAG GCC AAA AATCCC 804 Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Lys Asn Pro170 175 180 CCT GGA GAC TCC AGC CTA CAC TGG GCA GCC ATG GCA TTG CCA GCATTG 852 Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala Leu185 190 195 TTT TCT CTT ATA ATT GGC TTT GCT TTT GGA GCC TTA TAC TGG AAGAAG 900 Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys200 205 210 AGA CAG CCA AGT CTT ACA AGG GCA GTT GAA AAT ATA CAA ATT AATGAA 948 Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu215 220 225 230 GAG GAT AAT GAG ATA AGT ATG TTG CAA GAG AAA GAG AGA GAGTTT CAA 996 Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu PheGln 235 240 245 GAA GTG TAATTGTGGC TTGTATCAAC ACTGTTACTT TCGTACATTGGCTGGTAACA 1052 Glu Val GTTCATGTTT GCTTCATAAA TGAAGCAGCT TTAAACAAATTCATATTCTG TCTGGAGTGA 1112 CAGACCACAT CTTTATCTGT TCTTGCTACC CATGACTTTATATGGATGAT TCAGAAATTG 1172 GAACAGAATG TTTTACTGTG AAACTGGCAC TGAATTAATCATCTATAAAG AAGAACTTGC 1232 ATGGAGCAGG ACTCTATTTT AAGGACTGCG GGACTTGGGTCTCATTTAGA ACTTGCAGCT 1292 GATGTTGGAA GAGAAAGCAC GTGTCTCAGA CTGCATGTACCATTTGCATG GCTCCAGAAA 1352 TGTCTAAATG CTGAAAAAAC ACCTAGCTTT ATTCTTCAGATACAAACTGC AG 1404 273 amino acids amino acid linear protein unknown 61Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu -25 -20-15 -10 Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg-5 1 5 Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro10 15 20 Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu25 30 35 Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser40 45 50 55 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly LeuSer 60 65 70 Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp LeuVal 75 80 85 Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser PheLys 90 95 100 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe ArgIle Phe 105 110 115 Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val AlaSer Glu Thr 120 125 130 135 Ser Asp Cys Val Val Ser Ser Thr Leu Ser ProGlu Lys Asp Ser Arg 140 145 150 Val Ser Val Thr Lys Pro Phe Met Leu ProPro Val Ala Ala Ser Ser 155 160 165 Leu Arg Asn Asp Ser Ser Ser Ser AsnArg Lys Ala Lys Asn Pro Pro 170 175 180 Gly Asp Ser Ser Leu His Trp AlaAla Met Ala Leu Pro Ala Leu Phe 185 190 195 Ser Leu Ile Ile Gly Phe AlaPhe Gly Ala Leu Tyr Trp Lys Lys Arg 200 205 210 215 Gln Pro Ser Leu ThrArg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu 220 225 230 Asp Asn Glu IleSer Met Leu Gln Glu Lys Glu Arg Glu Phe Gln Glu 235 240 245 Val 1088base pairs nucleic acid single linear protein unknown CDS 151..885mat_peptide 226..885 62 AGCAGGGACA GTGGAGAGGG CGCTGCGCTC GGGCTACCCAATGCGTGGAC TATCTGCCGC 60 CGCTGTTCGT GCAATATGCT GGAGCTCCAG AACAGCTAAACGGAGTCGCC ACACCACTGT 120 TTGTGCTGGA TCGCAGCGCT GCCTTTCCTT ATG AAG AAGACA CAA ACT TGG ATT 174 Met Lys Lys Thr Gln Thr Trp Ile -25 -20 CTC ACTTGC ATT TAT CTT CAG CTG CTC CTA TTT AAT CCT CTC GTC AAA 222 Leu Thr CysIle Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lys -15 -10 -5 ACT GAAGGG ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC 270 Thr Glu GlyIle Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val 1 5 10 15 ACT AAATTG GTG GCA AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA 318 Thr Lys LeuVal Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys 20 25 30 TAT GTC CCCGGG ATG GAT GTT TTG CCA AGT CAT TGT TGG ATA AGC GAG 366 Tyr Val Pro GlyMet Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu 35 40 45 ATG GTA GTA CAATTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT 414 Met Val Val Gln LeuSer Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 50 55 60 TCA AAT ATT TCT GAAGGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT 462 Ser Asn Ile Ser Glu GlyLeu Ser Asn Tyr Ser Ile Ile Asp Lys Leu 65 70 75 GTG AAT ATA GTG GAT GACCTT GTG GAG TGC GTG AAA GAA AAC TCA TCT 510 Val Asn Ile Val Asp Asp LeuVal Glu Cys Val Lys Glu Asn Ser Ser 80 85 90 95 AAG GAT CTA AAA AAA TCATTC AAG AGC CCA GAA CCC AGG CTC TTT ACT 558 Lys Asp Leu Lys Lys Ser PheLys Ser Pro Glu Pro Arg Leu Phe Thr 100 105 110 CCT GAA GAA TTC TTT AGAATT TTT AAT AGA TCC ATT GAT GCC TTC AAG 606 Pro Glu Glu Phe Phe Arg IlePhe Asn Arg Ser Ile Asp Ala Phe Lys 115 120 125 GAC TTT GTA GTG GCA TCTGAA ACT AGT GAT TGT GTG GTT TCT TCA ACA 654 Asp Phe Val Val Ala Ser GluThr Ser Asp Cys Val Val Ser Ser Thr 130 135 140 TTA AGT CCT GAG AAA GGGAAG GCC AAA AAT CCC CCT GGA GAC TCC AGC 702 Leu Ser Pro Glu Lys Gly LysAla Lys Asn Pro Pro Gly Asp Ser Ser 145 150 155 CTA CAC TGG GCA GCC ATGGCA TTG CCA GCA TTG TTT TCT CTT ATA ATT 750 Leu His Trp Ala Ala Met AlaLeu Pro Ala Leu Phe Ser Leu Ile Ile 160 165 170 175 GGC TTT GCT TTT GGAGCC TTA TAC TGG AAG AAG AGA CAG CCA AGT CTT 798 Gly Phe Ala Phe Gly AlaLeu Tyr Trp Lys Lys Arg Gln Pro Ser Leu 180 185 190 ACA AGG GCA GTT GAAAAT ATA CAA ATT AAT GAA GAG GAT AAT GAG ATA 846 Thr Arg Ala Val Glu AsnIle Gln Ile Asn Glu Glu Asp Asn Glu Ile 195 200 205 AGT ATG TTG CAA GAGAAA GAG AGA GAG TTT CAA GAA GTG TAATTGTGGC 895 Ser Met Leu Gln Glu LysGlu Arg Glu Phe Gln Glu Val 210 215 220 TTGTATCAAC ACTGTTACTT TCGTACATTGGCTGGTAACA GTTCATGTTT GCTTCATAAA 955 TGAAGCAGCT TTAAACAAAT TCATATTCTGTCTGGAGTGA CAGACCACAT CTTTATCTGT 1015 TCTTGCTACC CATGACTTTA TATGGATGATTCAGAAATTG GAACAGAATG TTTTACTGTG 1075 AAACTGGCAC TGA 1088 245 aminoacids amino acid linear protein unknown 63 Met Lys Lys Thr Gln Thr TrpIle Leu Thr Cys Ile Tyr Leu Gln Leu -25 -20 -15 -10 Leu Leu Phe Asn ProLeu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg -5 1 5 Val Thr Asn Asn ValLys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 10 15 20 Lys Asp Tyr Met IleThr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 25 30 35 Pro Ser His Cys TrpIle Ser Glu Met Val Val Gln Leu Ser Asp Ser 40 45 50 55 Leu Thr Asp LeuLeu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 60 65 70 Asn Tyr Ser IleIle Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 75 80 85 Glu Cys Val LysGlu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 90 95 100 Ser Pro GluPro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe 105 110 115 Asn ArgSer Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr 120 125 130 135Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Gly Lys Ala 140 145150 Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu 155160 165 Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr170 175 180 Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn IleGln 185 190 195 Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu LysGlu Arg 200 205 210 215 Glu Phe Gln Glu Val 220 47 amino acids aminoacid single linear protein unknown 64 Glu Glu Ile Cys Arg Asn Pro ValThr Asp Asn Val Lys Asp Ile Thr 1 5 10 15 Lys Leu Val Ala Asn Leu ProAsn Asp Tyr Met Ile Thr Leu Asn Tyr 20 25 30 Val Ala Gly Met Asp Val LeuPro Ser His Xaa Trp Leu Arg Asp 35 40 45 10 amino acids amino acidsingle linear peptide unknown 65 Ile Thr Thr Leu Asn Tyr Val Ala Gly Met1 5 10 26 amino acids amino acid single linear peptide unknown 66 ValAla Ser Asp Thr Ser Asp Cys Val Leu Ser Xaa Xaa Leu Gly Pro 1 5 10 15Glu Lys Asp Ser Arg Val Ser Val Xaa Lys 20 25 12 amino acids amino acidsingle linear peptide unknown 67 Asp Val Leu Pro Ser His Cys Trp Leu ArgAsp Met 1 5 10 33 amino acids amino acid single linear peptide unknown68 Glu Glu Asn Ala Pro Lys Asn Val Glu Ser Leu Lys Lys Pro Thr Arg 1 510 15 Asn Phe Thr Pro Glu Glu Phe Phe Ser Ile Phe Asp Arg Ser Ile Asp 2025 30 Ala 9 amino acids amino acid single linear peptide unknown 69 GluSer Leu Lys Lys Pro Glu Thr Arg 1 5 5 amino acids amino acid singlelinear peptide unknown 70 Val Ser Val Xaa Lys 1 5 15 amino acids aminoacid single linear peptide unknown 71 Ile Val Asp Asp Leu Val Ala AlaMet Glu Glu Asn Ala Pro Lys 1 5 10 15 13 amino acids amino acid singlelinear peptide unknown 72 Asn Phe Thr Pro Glu Glu Phe Phe Ser Ile PheXaa Arg 1 5 10 29 amino acids amino acid single linear peptide unknown73 Leu Val Ala Asn Leu Pro Asn Asp Tyr Met Ile Thr Leu Asn Tyr Val 1 510 15 Ala Gly Asp Asp Val Leu Pro Ser His Cys Trp Leu Arg 20 25 24 aminoacids amino acid single linear peptide unknown 74 Ser Ile Asp Ala PheLys Asp Phe Met Val Ala Ser Asp Thr Ser Asp 1 5 10 15 Cys Val Leu SerXaa Xaa Leu Gly 20 22 amino acids amino acid single linear peptideunknown 75 Glu Ser Leu Lys Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu GluPhe 1 5 10 15 Phe Ser Ile Phe Xaa Arg 20 22 amino acids amino acidsingle linear peptide unknown 76 Glu Ser Leu Lys Lys Pro Glu Thr Arg AsnPhe Thr Pro Glu Glu Phe 1 5 10 15 Phe Ser Ile Phe Asp Arg 20 8 aminoacids amino acid single linear peptide unknown 77 Asn Ala Pro Lys AsnVal Lys Glu 1 5 16 amino acids amino acid single linear peptide unknown78 Ser Arg Val Ser Val Xaa Lys Pro Phe Met Leu Pro Pro Val Ala Ala 1 510 15 34 amino acids amino acid single linear peptide unknown 79 Ser LeuLys Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu Glu Phe Phe 1 5 10 15 SerIle Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Met Val Ala 20 25 30 SerAsp 37 amino acids amino acid single linear peptide unknown 80 Ser LeuLys Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu Glu Phe Phe 1 5 10 15 SerIle Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Met Val Ala 20 25 30 SerAsp Thr Ser Asp 35 40 amino acids amino acid single linear proteinunknown 81 Leu Arg Asp Met Val Thr His Leu Ser Val Ser Leu Thr Thr LeuLeu 1 5 10 15 Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr SerIle Ile 20 25 30 Asp Lys Leu Gly Lys Ile Val Asp 35 40 16 amino acidsamino acid single linear peptide unknown 82 Ser Arg Val Ser Val Thr LysPro Phe Met Leu Pro Pro Val Ala Ala 1 5 10 15 4 amino acids amino acidsingle linear peptide unknown 83 Pro Val Ala Ala 1 21 base pairs nucleicacid single linear DNA unknown 84 CCTGAGAAAG ATTCCAGAGTC 21 19 basepairs nucleic acid single linear DNA unknown 85 CTGCAGTTTG TATCTGAAG 1919 base pairs nucleic acid single linear DNA unknown 86 CATATAAAGTCATGGGTAG 19 27 base pairs nucleic acid single linear DNA unknown 87ACTTGTGTCT TCTTCATAAG GAAAGGC 27 21 base pairs nucleic acid singlelinear DNA unknown 88 TGTACGAAAG TAACAGTGTT G 21 22 base pairs nucleicacid single linear DNA unknown 89 ACTGCTCCTA TTTAATCCTC TC 22 23 basepairs nucleic acid single linear DNA unknown 90 CACTGACTCT GGAATCTTTCTCA 23 15 base pairs nucleic acid single linear DNA unknown 91TCGACCCGGA TCCCC 15 15 base pairs nucleic acid single linear DNA unknown92 TCGAGGGGAT CCGGG 15 25 base pairs nucleic acid single linear DNAunknown 93 TCTTCTTCAT GGCGGCGGCA AGCTT 25 18 amino acids amino acidsingle linear protein unknown 94 Asp Ser Arg Val Ser Val Xaa Lys Pro PhePhe Met Leu Pro Pro Val 1 5 10 15 Ala Ala 18 amino acids amino acidsingle linear protein unknown 95 Asp Ser Arg Val Ser Val Thr Lys Pro PhePhe Met Leu Pro Pro Val 1 5 10 15 Ala Ala 55 base pairs nucleic acidsingle linear DNA unknown 96 CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGCGTTGGAATTC GGTAC 55 49 base pairs nucleic acid single linear DNA unknown97 CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 9 base pairsnucleic acid single linear DNA unknown 98 TATGCAGGA 9 11 base pairsnucleic acid single linear DNA unknown 99 GATCTCCTGC A 11 17 base pairsnucleic acid single linear DNA unknown 100 TATGGAAGGT ATCTGCA 17 11 basepairs nucleic acid single linear DNA unknown 101 GATACCTTCC A 11 10 basepairs nucleic acid single linear DNA unknown 102 TTTCCTTATG 10 12 basepairs nucleic acid single linear DNA unknown 103 GCCGCCGCCA TG 12 25base pairs nucleic acid single linear DNA unknown 104 TCTTCTTCATGGCGGCGGCA AGCTT 25

What is claimed is:
 1. An isolated and purified non-human, mammalianstem cell factor polypeptide possessing hematopoietic biologicalactivity wherein said hematopoietic biological activity is thestimulation of the growth of early hematopoietic progenitor cells, saidpolypeptide selected from the group consisting of the polypeptides setforth in SEQ ID NOs: 50, 51, 52, 53 and
 55. 2. An isolated and purifiedrat stem cell factor polypeptide having the sequence of SEQ ID NO: 42.3. A rat stem cell factor polypeptide of claim 2 having the sequencedefined by the amino acid residues 1 to 162 of Seq Id No: 42, optionallyconsisting of an additional methionine residue at the N-terminus.
 4. Arat stem cell factor polypeptide of claim 2 having the sequence definedby the amino acid residues 1 to 164 of Seq Id No: 42, optionallyconsisting of an additional methionine residue at the N-terminus.
 5. Arat stem cell factor polypeptide of claim 2 having the sequence definedby the amino acid residues 1 to 165 of Seq Id No: 42, optionallyconsisting of an additional methionine residue at the N-terminus.
 6. Arat stem cell factor polypeptide of claim 2 having the sequence definedby the amino acid residues 1 to 193 of Seq Id No: 42, optionallyconsisting of an additional methionine residue at the N-terminus.
 7. Apharmaceutical composition comprising a hematopoietically effectiveamount of a non-human, mammalian stem cell factor polypeptide of claims1, 2, 3, 4, 5 or 6 and a pharmaceutically acceptable diluent, adjuvantor carrier.
 8. The pharmaceutical composition of claim 7 furthercomprising EPO, G-CSF, GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IGF-1 or IGF-2.
 9. The stem cellfactor polypeptides of claims 1, 2, 3, 4, 5 or 6 wherein said stem cellfactor polypeptide is conjugated to a water soluble polymer.
 10. Thestem cell factor polypeptide of claim 7 wherein said water solublepolymer is polyethylene glycol.
 11. A pharmaceutical compositioncomprising a hematopoietically effective amount of a non-human,mammalian stem cell factor polypeptide of claim 9 and a pharmaceuticallyacceptable diluent, adjuvant or carrier.
 12. The pharmaceuticalcomposition of claim 9 further comprising EPO, G-CSF, GM-CSF, CSF-1,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IGF-1 or IGF-2.